James E. Mace*, Barry J. Wilk and David A. Agard. Introduction. JMB MS 667 Cust. Ref. No. FC029/95

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1 Cust. Ref. No. FC029/95 [SGML] J. Mol. Biol. (1995) 251, Functional Linkage Between the Active Site of -Lytic Protease and Distant Regions of Structure: Scanning Alanine Mutagenesis of a Surface Loop Affects Activity and Substrate Specificity James E. Mace*, Barry J. Wilk and David A. Agard Howard Hughes Medical Institute and Department of Biochemistry and Biophysics University of California San Franciso, CA , USA *Corresponding author Previous structural and kinetic characterization of mutations within the active site of -lytic protease have demonstrated that amino acid residues in direct contact with the substrate are major substrate specificity determinants. The experiments described here identify residues of -lytic protease as a region of structure peripheral to the active site that also plays an important role in establishing the substrate specificity of the enzyme. Alanine substitution mutations within this surface loop of 19 amino acid residues significantly perturb the enzyme s specificity profile, despite being as far as 21 Å from the hydroxyl group of Ser195. The kinetic consequences of the mutations are remarkably independent of position within the loop and suggest that active site plasticity is affected more than static structure. Kinetic characterization of double mutants with the Met190 Ala broad-specificity active site mutation reveals varying degrees of nonadditivity and indicates that active site plasticity can be influenced through multiple sets of interactions. Although these results clearly demonstrate that tuning of serine protease activity is possible through remodelling of structure surrounding the active site, practical issues such as retaining compatibility with the folding mechanism and stability of the mature enzyme present significant obstacles to general application of the technique Academic Press Limited Keywords: protein engineering; substrate specificity; protein dynamics; scanning alanine mutagenesis; -lytic protease Introduction The primary function of an enzyme is to catalyze a chemical reaction by binding substrate(s) in a manner that stabilizes the reaction s transition state. The exact method by which this stabilization is achieved varies from enzyme to enzyme, but is always the result of tradeoffs between energetically favorable (e.g. hydrogen bonding and burial of hydrophobic surfaces) and unfavorable (e.g. bond distortion, cavity formation and entropic penalties) interactions. A key feature of the transition state complex is the precise positioning of the substrate molecule relative to the other components of the active site, a non-trivial requirement for molecules undergoing thermal fluctuations and other dynamic processes. Substrate must be held in place long Abbreviations used: LP, -lytic protease; IPTG, isopropyl- -D-thiogalactopyranoside; saap-, succinyl-l-ala-l-ala-l-pro-; pna, p-nitroaniline. enough for catalysis to occur, yet the interaction must be sufficiently weak to permit dissociation of the reaction products. Specificity results from an active site s ability to accommodate a limited range of possible transition state conformations and, hence, a restricted set of substrates (Fersht, 1985). Detailed understanding of the structural basis of substrate specificity is extremely valuable, both for the information it can provide concerning the nature and magnitude of the forces involved in catalysis and because it can permit rational modification of enzymes to produce desired changes in specificity. Determinants of substrate specificity may be probed by examining the kinetic consequences of systematic modification of substrate and enzyme structure. This approach has been used successfully for many systems including alcohol dehydrogenase (Murali & Creaser, 1986), carboxypeptidase Y (Mortensen et al., 1990), tyrosyl-trna synthetase (Fersht et al., 1985; Leatherbarrow et al., 1985), subtilisin (Wells et al., 1987a,b), glutathione reductase /95/ $08.00/ Academic Press Limited

2 Distant Mutations Affect Protease Substrate Specificity 117 (Scrutton & Perham, 1990) and trypsin (Craik et al., 1985). These studies also illustrated that thorough understanding of the interactions contributing to the specificity profile requires structural information at atomic resolution. -Lytic protease ( LP), an extracellular serine protease of Lysobacter enzymogenes homologous to mammalian serine proteases of the trypsin family (Whitaker, 1970; Silen et al., 1989), is a well-characterized system that can exhibit unique modes of substrate accommodation involving greatly increased active site plasticity while retaining high levels of activity (Bone et al., 1989b). It is well-suited for use in a detailed investigation of the relative importance of static and dynamic contributions to substrate specificity and to catalysis in general. The primary specificity (S 1 ) pocket of LP is defined by the side-chains of Met190, Met123 and Val218 and the polypeptide backbone of residues 214 to 216. Wild-type LP is specific for peptide substrates with small hydrophobic side-chains at the P 1 position, largely due to the two methionine side-chains at positions 190 and 213, which occupy most of the S 1 pocket. Mutation of either of these residues to alanine significantly broadens the specificity of the enzyme, enabling it to cleave substrates with large hydrophobic P 1 side-chains with high efficiency (Bone et al., 1991, 1989b). Analysis of X-ray structures of these mutants in complex with transition state analogues revealed that the active site had become significantly more plastic. Backbone shifts for residues 215, 216, 218, 219 and 219A, and side-chain repacking within residues 190, 213 and 218 permits the mutant enzymes to accommodate a wide range of P 1 side-chains. The backbone shifts resemble those observed for complexes of the same inhibitors with wild-type enzyme but are of considerably greater magnitude. The MA190 and MA213 analysis concluded that the observed patterns of substrate specificity arise from interactions between the substrate and the residues lining the specificity pocket, modulated indirectly by peripheral regions of the structure (Bone et al., 1991). One peripheral region expected to modulate the specificity of LP is the 19 amino acid residue long surface loop consisting of residues (see Figure 1). The two segments of polypeptide backbone with greatest plasticity in the broadly specific LP mutants are either adjacent (residues The P and S nomenclature of Schechter & Berger (1967) is used. P 1 is the substrate residue before the scissile bond; P 2, P 3, etc extend toward the N terminus. S 1, S 2, etc are the corresponding binding subsites on the enzyme. Residues are numbered according to Fujinaga et al. (1985) and reflect structural homology with chymotrypsin. Previous papers from the Agard lab used a slightly different numbering scheme (James et al., 1978). For reference, Met192 in the old scheme becomes 190, Val217A becomes 218 and Met213 remains 213. Figure 1. Diagram of the surface loop and active site region of LP. The loop C backbone and side-chains are shown in ball-and-stick form. Key active site sidechains are shown as space-filling spheres, and the remainder of the molecule is displayed in ribbon form. This figure was prepared using MOLSCRIPT and RASTER3D (Kraulis, 1991; Merritt & Murphy, 1994) ) or contained within (residues A) this loop (Bone et al., 1991). Residues A are found to have significant plasticity in the wild-type enzyme as well, adopting different conformations when a variety of transition state analogue inhibitors are bound (Bone et al., 1989a). An extensive network of hydrogen bonds and van der Waals contacts both within the loop and with key active site residues may be one mechanism by which the active site conformation is maintained and its flexibility constrained. Examination of the structures of related serine proteases reveals that although the sequence of this loop is quite variable, it is highly conserved structurally. In the five related proteases aligned in Figure 2, there is a conserved disulfide bond involving Cys220A, and the relative positions of the ends of the loop (residues 216 and 226) are essentially identical since, in all cases, the loop connects two antiparallel strands which form one wall of the S 1 pocket. However, in each of these enzymes, the loop is considerably shorter than in -lytic protease. The increased length of the loop in LP may contribute to the remarkable plasticity of its active site. Additional evidence for the importance of this region comes from attempts to alter the substrate specificity of trypsin. Although chymotrypsin-like specificity could be conferred upon trypsin by modifying amino acid residues in direct contact with

3 118 Distant Mutations Affect Protease Substrate Specificity substrate, the sequence and hence structure of residues was found to be crucial for efficient hydrolysis of amide substrates (Hedstrom et al., 1992). The conformation and physical properties of this surface loop clearly contribute to the structure and function of the active site. The dependence of enzymatic activity upon peripheral structural elements is by no means unique to serine proteases. However, since the majority of directed mutagenesis experiments target residues in direct contact with substrate of which are vital for catalysis, discovery of such dependencies is often fortuitous or a byproduct of investigations of the active site proper, as was the case for LP. In systems where distant mutations have been found to affect catalysis, the effects are frequently the result of changes to domain or subunit interfaces. Mutations at the domain interface of papain, 15 Å from the active site, have been found to significantly affect k cat without detectably altering active site geometry (Altschuh et al., 1994). Similar long-range effects for mutations at interfaces in -amylase have also been observed (Holm et al., 1990). In other cases, structural perturbations are propagated within single domains. Recent examples from the literature include bacteriophage T4 lysozyme (Hurley et al., 1992) and subtilisin E (Chen & Arnold, 1993). Generally, such peripheral mutations are assumed to generate static structural perturbations. Alternatively, they might affect the flexibility of the enzyme, thereby reducing access to modes essential for catalysis. Proteins are dynamic systems and flexibility is often intimately associated with function, as has been demonstrated for glutathione synthase (Tanaka et al., 1993), citrate synthase (Zhi et al., 1991), porcine pancreatic phospholipase A 2 (Kuipers et al., 1991) and a variety of other allosteric and non-allosteric enzymes. To better understand how the surface loop of LP might modulate substrate specificity, the loop has been replaced with the homologous portions of trypsin, chymotrypsin, elastase, and proteases A and B of Streptomyces griseus. In addition, scanning alanine mutagenesis has been employed to probe the structure of the loop in a less disruptive fashion. In each case, mutations were tested in the context of both the wild-type active site and the MA190 broad-specificity mutant. Variations in production levels, precursor processing and kinetic properties of the enzymes upon a variety of substrates were examined. Structural information for LP was used in the interpretation of the resulting biochemical data. Due to the large number of insertion residues in the LP sequence, the notation used for mutations is slightly non-standard, with the substitution represented by 1-letter amino acid codes in the first two characters and the position of the mutation by the remaining numbers and letters. Hence, SA219A represents an alanine substitution for serine 219A. Double mutations are indicated by a plus ( + ) symbol, as in GA216 + MA190. Results and Discussion Exchange of the surface loop The three-dimensional structures of the loops of LP, trypsin, chymotrypsin, elastase and S. griseus proteases A and B vary considerably, but by superimposing the backbone atoms of residues 216 and 226 it is possible to graphically graft each homologous loop onto LP. Significantly, it is also possible to model the conserved disulfide in each of the chimeric molecules with only major changes. Energy minimization calculations performed on a model structure of LP bearing the loop of S. griseus protease B indicated that reasonable structure should be retained in the S 1 pocket (data not shown). Given the success of computer modelling and the observed structural homology, it seemed reasonable to proceed with the mutagenesis experiments. It is known that LP requires its 166 amino acid residue pro region for proper folding (Silen et al., 1989). The pro region is also a potent competitive inhibitor of the mature enzyme with an inhibition constant (K I ) of approximately M (Baker et al., 1992a). These and other lines of evidence suggest that interactions exist between the pro and mature regions in the vicinity of the active site. It was anticipated that large perturbations of the surface structure of LP, such as exchange of the surface loop with the homologous regions of related serine proteases, might interfere with proper expression. Were the loop swap to produce a significant alteration in the substrate specificity of activity, the mutant protease might also not be able to proteolytically process the covalently attached (cis) pro region in the appropriate manner. The mature domain of LP, which does not fold independently, can be folded by its pro region either in vitro or in vivo when the two polypeptides are expressed separately (in trans) (Baker et al., 1992b; Silen & Agard, 1989). To maximize the likelihood of obtaining properly folded but not necessarily catalytically active material, loop swap mutants were constructed in combination with both wild-type (narrow specificity) and MA190 (broad specificity, high plasticity) active sites, and in both cis (pcomp10) and trans (palp11) expression vectors. When cells carrying each of these constructs were grown in culture, no active protease was detectable in either the culture supernatant or cell lysate. For each chimeric enzyme, expression of alkaline phosphatase was detected, indicating that the PhoA promoter driving LP expression had been induced by phosphate depletion in the cultures. Western blots of lysed cells revealed ladders of degradation products extending down from precursor-size material (data not shown), strongly suggesting that the mutant proteins were not folding properly. The absence of a significant band the size of the mature enzyme may indicate that the folded form is extremely unstable. Western blots of culture media

4 Distant Mutations Affect Protease Substrate Specificity 119 Figure 2. The loop of LP shows limited sequence homology with the corresponding region of Streptomyces griseus proteases A and B, bovine chymotrypsin and trypsin and porcine elastase; however, it is considerable longer. showed that neither mature-sized protease nor fragments were present. Since secretion of LP has been shown to require proper folding (Fujishige et al., 1992), the absence of LP fragments in the media provides additional evidence that the protease never folds successfully. The shorter loops from the homologus proteases are apparently incompatible with either the folded form of the enzyme, block pro region binding, or interfere with stabilization of some intermediate state in the folding pathway. Effects of alanine substitutions on enzyme production Scanning alanine mutagenesis has proven to be a powerful method for examining the interplay between structure and function. Side-chains beyond the carbon atom can be eliminated without significantly altering the main-chain conformation (Cunningham and Wells, 1989). Given the apparent involvement of the loop in the folding process of LP, this less disruptive approach to mutagenesis was expected to be more successful. Scanning alanine mutagenesis will selectively disrupt hydrogen bonds involving side-chains of the loop. However, due to the compact nature of the loop, the same alanine substitutions will also introduce subtle structural perturbations involving minor backbone shifts and side-chain repacking. The 18 non-alanine residues in the surface loop of LP were replaced, in turn, with alanine to investigate the effects of these perturbations on production and activity. All scanning alanine mutations were made in palp12 cis expression vectors in the context of both the wild-type and MA190 active sites. Alanine substitutions in the loop had major effects on enzyme production (Table 1). Secreted levels of wild-type and MA190 -lytic protease typically reached 1800 nm for control cultures. Production levels for the scanning alanine mutants ranged from normal (e.g. SA219A and SA219A + MA190) to levels undetectable (<0.05 nm) by the ELISA protocol employed for concentration determination (e.g. NA220 and CA220A). Following purification, the mutants were resistant to autoproteolysis at 0 C and 25 C, suggesting that the reduced production levels are due to defects in the folding pathway rather than gross instability of the folded enzyme. Processing of the precursor during folding involves cleavage at a threonine residue between the pro and mature regions. Kinetic characterization (described below) of those mutants expressed at detectable levels revealed that all, with the possible exception of GA216, retained significant activity toward threonine substrates, implying that they should all be able to process themselves with the pro region in cis. The conclusion, therefore, is that the pro region catalyzed folding of LP appears to be exquisitely sensitive to structure in the vicinity of the surface loop. Even the minor perturbations introduced by scanning alanine mutagenesis are sufficient to interfere with production levels of mature enzyme. All amino acid residues within the loop expose some backbone or side-chain atoms to Table 1. Scanning alanine mutations within the loop have significant effects on protein production Approximate LP concentration in culture supernatant (nm) Wild-type MA190 Mutation active site active site GA NA VA QA SA219A NA219B GA219C NA219D NA220 n.d. n.d. CA220A n.d GA IA PA222A SA222C QA RA SA SA ELISA assays of protease purified from culture supernatants were performed to determine enzyme concentration. Although variation in expression for individual isolates of the various mutants was observed, it was less than the order-of-magnitude differences between mutants. Activity measurements against succinyl-ala-pro-ala-p-nitroalanine substrate before and after purification were used to back-calculate an approximate concentration for the protease in the original culture supernatant. n.d., not detectable.

5 120 Distant Mutations Affect Protease Substrate Specificity solvent. One explanation for the altered production levels is that they alter the surface structure of the mature protease. However, no direct correlation exists between accessible surface area and altered production levels. Instead it appears that these residues act in concert to determine the shape and chemical characteristics of part of the pro region binding interface. Small changes may be sufficient to result in misregistration of the pro and mature regions or otherwise adversely affect the folding reaction, resulting in decreased yields of stable protein. In contrast, Asn220, Cys220A and Arg224 are largely buried, yet substitution with alanine at these positions abolishes production of LP. The side-chains of these residues participate in important structural interactions and disruption of these interactions is likely to cause significant changes within the loop and adjacent structural elements (see Figure 1). Asn220 is part of a hydrogen bond network involving residues 185, 190, 217 and 226; Cys220A is linked through a disulfide bond to Cys189; the polar atoms of the side-chain of Arg224 make hydrogen bonds with residues 220, 220A and 222, while the non-polar atoms form a scaffold which supports the upper lobe (residues D) of the loop. The observed differences in expression levels for the same alanine substitution in conjunction with either a wild-type or an MA190 active site are intriguing and suggest a significant degree of structural interplay between the surface loop and active site residues in the vicinity of Met190. Crystallographic analysis of MA190 versus wild-type LP has revealed only extremely minor structural changes in the surface loop, concentrated mainly around Gly216 (Bone et al., 1989b). However, as discussed above, even minor perturbations in the loop can have major effects on production. Many loop residues make van der Waals contacts or hydrogen bonds with structural elements adjacent to Met190. For these residues, expression levels vary depending on whether residue 190 is Met or Ala. However, there are a number of positions within the loop (e.g ) which do not directly interact with the region around 190, yet still show 190-dependent expression levels. Clearly, structural perturbations are being conducted over large distances ( 10 Å) through the protein matrix. This behavior is not surprising given the compact nature of the loop and the large number of interactions with the active site of the enzyme. Effects of alanine substitutions on substrate specificity If the loop does in fact modulate activity and specificity, these perturbations should measurably affect the kinetics of the enzyme. The kinetic properties of each scanning alanine mutant produced at levels above 2 nm in the culture medium were characterized using a panel of substrates of the form succinyl-l-ala-l-ala-l-pro-x-p-nitroaniline (saapx-pna), where X is Gly, Ala, Val, Leu, Ile, Met, Phe, or Thr (see Table 2). The first seven of these substrates form a hydrophobic series with P 1 side-chains of increasing size and provides a fairly rigorous test for altered properties of the S 1 pocket. For the simple cleavage of peptide substrates by a serine protease, the kinetic parameter k cat /K M is the effective second-order rate constant for the reaction. Combining effects due to substrate binding and transition state stabilization, this parameter is useful for assessing altered substrate specificity. It can be shown from transition state theory that the amount of transition state stabilization achieved for a particular combination of enzyme and substrate is given by RTln(k cat /K M ), where R is the gas constant and T is the absolute temperature, both in the appropriate units (Fersht, 1985). Examination of the plots in Figure 3 reveals that significant changes in k cat /K M are obtained for the various scanning alanine mutants on the eight substrates. With one notable exception, GA216, the scanning alanine mutants possess substrate specificity profiles that are modulated versions of their parent enzyme s. Good substrates for the wild-type (or MA190) enzyme continue to be good substrates in the scanning alanine mutants. The rank ordering of substrate preferences for the various mutants remains fairly constant, with some switching among substrates with similar values of k cat /K M (for example, between Phe and Met for MA190 active sites, or among Ile, Leu and Phe for wild-type active sites). There are, however, some interesting conclusions that can be drawn from an examination of these modulated specificities. With a wild-type active site, most alanine substitutions tend to lower activity against small substrates and raise activity against large substrates. This would be consistent with either increased plasticity in the active site (as observed crystallographically for the MA190 active site mutant of LP: Bone et al., 1989b) or a static expansion of the binding pocket as a result of the surface loop s accommodation of the alanine substitution. Coloration of a computer model of the loop based upon the magnitude (large versus small) of the perturbations to the specificity profiles results in a clear demarkation of shells of residues within the loop (see Figure 4). Mutation of internal residues participating in the -sheet that forms the left wall of the substrate binding cleft show greatly altered P 1 specificities. These residues are 216, 217, 225 and 226. Conversion of solvent-exposed residues along the upper lobe of the loop (218, 219 and 219D) and two residues within the lower lobe (222 and 222A) to alanine also produces large changes in substrate preference. The loop residues for which alanine substitution results in only small specificity changes (219A, 219B, 219C, 221, 222C and 223) are extremely solvent-exposed (side-chains directed outward) and occupy the outermost extremes of the loop. All of these amino acid residues are wrapped around a core of residues (220, 220A and 224) which appear to be important for maintaining a stable structure. From this analysis it may be seen that substitutions that

6 DEEP PAGE Distant Mutations Affect Protease Substrate Specificity 121 Table 2. Kinetic parameters and associated errors for alanine substitution mutations in the loop produced to levels of 3 nm or better in culture Enzyme Substrate k cat/k M (M 1 s 1 ) k cat(s 1 ) K M(mM) Wild-type Gly Ala 26, Val Leu Ile Met Phe Thr GA216 Gly Ala Val Leu Ile Met Phe Thr NA217 Gly Ala 12, Val Leu Ile Met 23, Phe Thr VA218 Gly Ala Val Leu Ile Met Phe Thr QA219 Gly Ala 36, Val Leu Ile Met 15, Phe Thr SA219A Gly Ala 10, Val Leu Ile Met Phe Thr NA219B Gly Ala 13, Val Leu Ile Met Phe Thr GA219C Gly Ala 12, Val Leu Ile Met Phe Thr NA219D Gly Ala 20, Val Leu Ile Met Phe Thr continued

7 122 Distant Mutations Affect Protease Substrate Specificity Table 2. continued Enzyme Substrate k cat/k M (M 1 s 1 ) k cat(s 1 ) K M(mM) GA221 Gly Ala 21, Val Leu Ile Met Phe Thr IA222 Gly Ala 48, Val Leu Ile Met 15, Phe Thr PA222A Gly Ala 74, Val Leu Ile Met Phe Thr SA222C Gly Ala 14, Val Leu Ile Met Phe Thr QA223 Gly Ala 19, Val Leu Ile Met Phe Thr SA225 Gly Ala Val Leu Ile Met Phe Thr SA226 Gly Ala 37, Val Leu Ile Met Phe Thr MA190 Gly Ala 13, Val Leu 56, Ile Met 406,000 63, Phe 222,000 16, Thr 41, GA216 + MA190 Gly Ala Val Leu Ile Met Phe Thr continued

8 Distant Mutations Affect Protease Substrate Specificity 123 Table 2. continued Enzyme Substrate k cat/k M (M 1 s 1 ) k cat(s 1 ) K M(mM) NA217 + MA190 Gly Ala Val Leu 70, Ile Met 124,000 10, Phe 115, Thr VA218 + MA190 Gly Ala Val Leu 39, Ile Met 153,000 16, Phe 202,000 32, Thr QA219 + MA190 Gly Ala Val Leu 19, Ile Met 27, Phe 25, Thr SA219A + MA190 Gly Ala Val Leu 16, Ile Met 24, Phe 20, Thr NA219B + MA190 Gly Ala Val Leu 27, Ile Met 46, Phe 69, Thr 11, NA219D + MA190 Gly Ala Val Leu 15, Ile Met 58, Phe 77, Thr GA221 + MA190 Gly Ala Val Leu Ile Met 19, Phe 21, Thr IA222 + MA190 Gly Ala Val Leu 22, Ile Met 38, Phe 71, Thr PA222A + MA190 Gly Ala Val Leu 17, Ile Met 28, Phe 29, Thr continued

9 124 Distant Mutations Affect Protease Substrate Specificity Table 2. continued Enzyme Substrate k cat/k M (M 1 s 1 ) k cat(s 1 ) K M(mM) SA222C + MA190 Gly Ala 12, Val Leu 112,000 25, Ile Met 184,000 41, Phe 244,000 55, Thr 47,900 12, QA223 + MA190 Gly Ala Val Leu 136,000 14, Ile Met 251,000 23, Phe 287,000 26, Thr 37, RA224 + MA190 Gly Ala Val Leu 77,400 10, Ile Met 117, Phe 125, Thr 15, SA225 + MA190 Gly Ala Val Leu 81, Ile Met 162,000 22, Phe 152,000 18, Thr 37, SA226 MA190 Gly Ala 11, Val Leu 127,000 27, Ile Met 251,000 56, Phe 279,000 54, Thr 44, Values of k cat/k M were determined from the slopes of double reciprocal plots of initial reaction velocity as a function of substrate concentration and values of k cat and K M from the intercepts. Error computation and propagation was carried out as described by Bevington (1969). Substrates were of the form succinyl-ala-ala-pro-x-p-nitroaniline, with the P 1 residue X one of Gly, Ala, Val, Leu, Ile, Met, Phe or Thr. All kinetic constants presented in this Table were obtained from measurement of reactions in microtiter plates as described in Experimental Methods with the exception of the Phe substrate on the wild-type enzyme which is the previously reported value (Bone et al., 1989b) determined in 1 cm path-length cuvettes on a standard spectrometer. should have minimal structural impact do indeed have minimal effects upon enzymatic activity. The observed alterations in the substrate specificity profiles are consistent, at least at a gross level, with the structural roles of the mutated residues. With an MA190 active site, perturbations of the substrate specificity profile are also apparent, but of a considerably different nature. They are manifest most often as a reduction in activity toward the full range of substrates. For NA217 + MA190 and VA218 + MA190, there appears to be some selective destabilization of transition states (lowered k cat /K M ) involving small P 1 side-chains similar to that observed for the same mutations in the context of a wild-type active site. The effects are more uniform for the other mutants, however the transition states for Leu, Ile and Phe substrates are consistently less destabilized than those for substrates with smaller P 1 side-chains. In only a few instances is there an increase in k cat /K M over that observed for MA190 LP. The mutations with least effect in the context of the wild-type active site can have greater influence upon the activity of the enzyme when combined with an MA190 active site. SA219A + MA190 and GA221 + MA190 show consistent three to tenfold reduction in activity for all substrates; SA222C + MA190 show only slight changes; GA219C + MA190 is defective in production and could not be characterised. It is therefore clear that LP responds differently to alanine substitutions in the context of the MA190 active site. k cat effects dominate the altered values of k cat /K M Although some care must be taken in interpretation as a result of propagated errors in the parameters, it is possible to dissect the altered values

10 Distant Mutations Affect Protease Substrate Specificity 125 Figure 3(a) and (b). (Legend page 126)

11 126 Distant Mutations Affect Protease Substrate Specificity of k cat /K M into contributions from k cat and K M. For both active sites (wild-type or MA190), the major, but by no means exclusive, source of the altered values of k cat /K M is k cat. These mutations affect the enzyme s ability to stabilize the transition state more than they affect its ability to bind substrate. In contrast, mutations within the S 1 pocket predominantly affect K M (Bone et al., 1991). For scanning alanine mutants with either wild-type or the MA190 active site, changes in K M for particular substrates are typically less than an order of magnitude; however, k cat shows significantly greater variation. For mutants with a wild-type active site, the range in k cat is, on average, 11 times greater than the range in K M. For MA190 active sites, the average range in k cat is only fourfold greater than the range in K M. Examination of the actual values of k cat and K M in Table 2 reveals that the lower ratio for MA190 active sites relative to wild-type is due to reduced variation in k cat and not increased variation in K M. A notable exception to the dominance of k cat effects over K M occurs for the Phe substrate on mutants with a wild-type active site for which the range in K M is tenfold greater than the range in k cat. saapf-pna is an extremely poor substrate for wild-type LP because the S 1 pocket is too small to easily accommodate the bulky Phe P 1 side-chain. The effects of the alanine substitution mutations upon the other substrates, whose P 1 side-chains reside properly within the S 1 pocket, are considerably different and warrant detailed analysis. Structural interpretation of altered specificity Given the large body of structural information that has been accumulated for wild-type and MA190 LP, both free and in complex with a variety of transition-state-analog inhibitors, it is possible to speculate upon the structural origins of the altered specificity of the scanning alanine mutants. Often in cases such as this, static structural perturbation within the active site is invoked to explain changes in substrate preference. For LP, however, both altered steric constraints within the substrate binding pocket and modified dynamic properties of the active site are important, as indicated by the highly plastic nature of the S 1 pocket in the MA190 enzyme and the remarkably similar effects of so many of the scanning alanine mutations within the surface loop. It is difficult to see how, for mutants with wild-type active sites, the consistent pattern of decreased activity toward substrates with small P 1 residues and increased activity toward those with larger P 1 side-chains might arise solely from static rearrangements within the S 1 pocket. The various alanine substitutions within the loop, far removed from the active site, would have to give rise to the same structural perturbations. On the other hand, were the mutations to affect the dynamic behavior of the active site by increasing its plasticity, the observed alterations in substrate specificity could be explained quite simply. The alanine substitutions remove side-chain atoms that potentially serve to stabilize the loop. It is not unreasonable to expect that such changes might result in an expanded set of accessible conformations for the loop and active site. In the discussion that follows, it is important to distinguish between plasticity and flexibility in the description of protein dynamics. We are using plasticity to refer to positional variation that is constrained to a set of local energy minima. Transition from one accessible conformation to another requires traversal of an energy barrier of some height. Flexibility refers to the more degenerate case with a continuum of possible conformation of equal energy. Structural analysis of MA190 LP and the relevant inhibitor complexes has provided strong evidence for the importance of conformational plasticity in the function of this enzyme (Bone et al., 1989b). In the MA190 active site, few steric constraints are placed upon the P 1 side-chain as a result of the deep S 1 pocket created by the removal of atoms from the side-chain of Met190 and the enhanced active site plasticity. The P 1 side-chain is effectively sandwiched between two sheets, one of which can bow inward or outward to accommodate it, allowing the enzyme to utilize the majority of the available favorable enthalpy arising from burial of hydrophobic surface area. Plasticity in the region of A permits improved packing and exclusion of solvent from the pocket. This lack of steric constraints within the S 1 pocket helps explain the broad specificity of the MA190 enzyme and makes it likely that the changes in k cat /K M for alanine substitution mutants within the loop of the MA190 mutant will be the result of altered dynamic properties rather than of static changes in active site geometry. In these mutants, the typical effect on substrate specificity is decreased activity toward the full range of substrates, exactly the sort of behavior expected for an active site possessing enhanced plasticity. The number of accessible conformations for the enzyme-substrate complex increases, resulting in a decreased reaction rate and lowered k cat /K M due to the increased entropy of the system that can be offset only partially by the limited amount of additional binding enthalpy available for larger P 1 side-chains. In wild-type LP, there is considerable substrate discrimination due to steric clash between the P 1 Figure 3. (a) Plot of log 10(k cat/k M) = log 10(k cat/k M) mutant log 10(k cat/k M) wild-type with associated errors for the 18 scanning alanine mutants bearing a wild-type active site. Values for k cat/k M are from Table 2. The quantity log 10(k cat/k M) mutant log 10(k cat/k M) wild-type is proportional to the relative amount of transition state stabilization achieved for a given substrate and mutant with respect to the wild-type enzyme. Negative values indicate reduced activity. Positive values indicate increased activity. (b) Same as (a), except that the quantity plotted is log 10(k cat/k M) mutant log 10(k cat/k M) MA190 for mutants bearing MA190 active sites.

12 Distant Mutations Affect Protease Substrate Specificity 127 side-chain of the substrate and the components of the S 1 pocket, principally Met190, Met213 and Val218. The pocket accommodates alanine and valine side-chains quite effectively although significant packing defects remain; larger side-chains fit poorly and force the substrate to twist outward along an axis defined by its sheet interactions with residues 215 and 216 of the enzyme. The S 1 site of LP is not an exact fit for any P 1 side-chain and K M values for substrates of the form saapx-pna are all remarkably similar and quite high (mm). Given this, it is possible to rationalize the altered specificity profiles of the scanning alanine mutants with wild-type active sites entirely in terms of altered active site dynamics. If, as was presumed to occur for the MA190 active site, the alanine substitutions slightly enhance active site plasticity, the resultant increased entropic penalty associated with accessing the catalytically competent conformation will yield a decreased reaction rate (lower k cat /K M ). Since the wild-type S 1 pocket does impose significant, although rather non-specific, steric constraints upon the P 1 side-chain, these entropic penalties could be offset by increased enthalpic contributions arising from the burial of additional hydrophobic surface area for larger side-chains facilitated by the enhanced dynamic properties of the active site. Enzyme-substrate complexes involving larger P 1 residues would be more highly constrained within the active site than those possessing smaller P 1 side-chains. Such rebalancing of entropic and enthalpic contributions would result in decreased activity toward substrates with small P 1 side-chains and an increase toward those with larger side-chains. Although dynamic arguments can explain many features of the altered specificity profiles for the scanning alanine mutants in the loop of LP, in reality the activity and substrate preference of the enzyme is determined by the combined and dynamic properties of the active site. Crystallographic analysis of two mutants within the loop, NV217 (J. F. Reidhaar-Olson, & D. A. Agard, unpublished results) and VA218 (R. Bone & D. A. Agard, unpublished results) has revealed significant enlargement of the S 1 pocket due to altered conformation of residues Static effects are therefore likely to provide the dominant contribution to changes in substrate preference for the NA217 and VA218 mutants. Examination of the changes in k cat /K M for NA217 + MA190 and VA218 + MA190 (Figure 3(b)) reveals anomalous P 1 dependencies, suggesting that static perturbations are overwhelming the ability of the plastic binding pocket to optimally accommodate smaller substrates. For other residues in the loop, dominance of static and dynamic effects will vary, being particularly sensitive to the nature of the wild-type versus MA190 S 1 pocket. Position 216 is a special case Substitution of alanine for glycine at position 216 is an exception to the observation that alanine substitution in the loop results in only minor specificity changes. With a wild-type active site, k cat /K M for those substrates for which kinetic characterization was possible (the GA216 enzyme was produced poorly) was affected by more than an order of magnitude. In contrast GA216 + MA190 LP was produced at reasonable levels and full kinetic characterization was possible. The k cat /K M for substrates with Phe at the P 1 position (the most preferred substrate for MA190 LP) decreased 4300-fold, requiring a 5.0 kcal/mol destabilization of the transition state. Such a large energetic difference for a single substrate indicates that there should exist an identifiable structural basis for the effect. Less severe perturbations to the specificity profile of GA216 + MA190 include relative decreases in activity toward Ala, Leu, Met and Thr. These observations are consistent with a P 1 pocket that has collapsed slightly and become more rigid. Residues form one of the two regions found to be most plastic in crystallographic studies of MA190 and wild-type LP. Although Gly216 does not occupy a forbidden region of - conformational space, the restricted backbone conformations for alanine, relative to glycine at position 216, should greatly limit the plasticity of this region and reduce the enzyme s ability to accommodate substrates with bulky P 1 side-chains in the active site. It should also limit the ability of the S 1 pocket to contract to fit small P 1 residues such as glycine. Since substrates make extended -strand-type interactions with residues 215 and 216, changes to the backbone geometry of this region of LP could have significant effects on the enzyme s ability to position the substrate for efficient catalysis. The fact that the enzyme is still highly active clearly indicates that the new backbone conformation is compatible with (although perhaps not optimal for) substrate binding and transition state stabilization. Similar results had previously been obtained for the Gly216 Ala mutation in rat pancreatic trypsin (Craik et al., 1985), indicating that residue 216 may be an excellent mutagenesis target for redesigning substrate specificity within the serine protease family. Computer modelling suggests that the side-chain of Ala216 can interact with substrate P 1 side-chains larger than alanine, so the observed kinetic profile is a combination of effects due to altered backbone geometry and dynamics, and to steric clash with the side-chain of residue 216. Distinguishing among these various contributions will require crystallographic analysis of complexes of GA216 + MA190 with peptide transition state analogs. Such an analysis for this and other mutations at position 216 in LP is underway and will be described separately. Functional interactions between loop residues and the active site The existence of altered specificity profiles for the majority of the scanning alanine mutants indicates that the activity of LP is modulated by distant regions of structure, with perturbations being

13 128 Distant Mutations Affect Protease Substrate Specificity Figure 4. Effects on substrate specificity of alanine substitutions in the surface loop of wild-type LP decrease with increasing distance from the active site. Loop residues are shown as space-filling spheres shaded light gray for large effects and dark gray for small effects. The three residues for which alanine substitution abolished LP production are colored black. The His57 side-chain is shown to indicate the location of the active site. This figure was prepared using MOLSCRIPT and RASTER3D (Kraulis, 1991; Merritt & Murphy, 1994). propagated through the protein matrix to reach the active site. Examination of the kinetic consequences of the various mutations reveals that the effects of multiple changes within the active site and loop are often non-additive, even when the changes are quite remote from one another. This provides direct and quantifiable evidence that a significant mass of protein surrounding the active site of LP is important for damping structural perturbation and governing the function of the enzyme. When a mutation is introduced into a protein, rebalancing of the atomic-level forces and entropic contributions to the stability of the molecule as a whole takes place (Mark & van Gunsteren, 1994). It may or may not be accompanied by detectable atomic rearrangements as a considerable amount of energy can be managed by minimal changes in charge distributions, bond angle, or bond lengths (Bone & Agard, 1991; Welch, 1986), and because entropy changes are not directly measurable through equilibrium techniques of structure determination such as crystallography. Stabilization may be accomplished within a small volume of the protein, especially for conservative mutations. However, changes within a larger portion of the protein matrix are likely to be required to accommodate a highly disruptive mutation. Overlap between the volumes required to stabilize multiple mutations causes the effects of the mutations to become non-additive. From the extensive body of kinetic information obtained for the scanning alanine mutations, it is possible to construct a set of double-mutant (thermodynamic) cycles from the values of RTln (k cat /K M ) (Figure 5(a) and Table 3). For each substrate a coupling energy G coup may be defined as Figure 5. (a) Definitions for components of the thermodynamic cycle and of G coup, the coupling energy, for scanning alanine mutations and Met190. G ts is the G computed from k cat/k M for a given substrate as described in the text. Values for these quantities are presented in Table 3. Enzymes are denoted by a combination of wt (wild-type active site), sa (scanning alanine mutation) and MA190 (MA190 active site). (b) Coupling energies between scanning alanine mutations in the loop and Met190 vary from residue to residue and are influenced to varying degrees by the P 1 amino acid residue of the substrate. For each position in the loop for which a coupling energy could be calculated in Table 3, the maximum and minimum values of G coup for the eight substrates are plotted as horizontal bars and the average value as a filled circle. G 2 ' G 2, which represents the difference in transition state stabilization associated with introducing a given alanine substitution in the context of an MA190 active site relative to a wild-type active site. Non-zero values of G coup indicate structural interdependence of the scanning alanine and MA190 mutations. Positive values indicate that an alanine substitution mutation stabilizes the transition state more in the presence of a wild-type active site. Equivalently, the mutation might be said to be less destabilizing for wild-type enzymes than for the corresponding MA190 variant. Negative values of G coup indicate preferential stabilization of the