In mice: V H ~250 D ~15 J ~5. L 1 -V H1 -L 2 -V H2 -L 3 -V H3 ---L n -V Hn ----D 1 -D 2 -D 3 ---D n ----J 1 -J J n V L ~250 J ~4

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Loren Hunter
6 years ago
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1 Lecture 14 ymes Cont. ymes preferentially stabilize transitions states versus ground states ite directed mutagenesis (Tyr ta synthetase) Transition state analogue inhibitors Design of protein active sites that stabilize the transition state CDs have large sequence variation (protypic diversity system) 1. multiple germline V genes light chain V, J; heavychain V, D, J In mice: V ~250 D ~15 J ~5 Antibodies V L 1 V 1 L 2 V 2 L 3 V 3 L n V n D 1 D 2 D 3 D n J 1 J 2 J n Antibody IgG light chain C L heavy chain 250 AAs C 1 C 2 C 3 constant region variable region (125 AAs) Binding site for antigen, consists of 6 hypervariable loops, 3 from light chain, 3 from heavy chain LV 1 D 2 J 1 V L ~250 J ~4 V L LV L1 LV n J 1 J 2 J n splice together in different combinations in mice ~20,000 different combinations (combinatorial diversity) 2. recombination inaccuracy When you combine D genes insertions and deletions can occur ften use smaller fragments: Fab fragment V scfv fragment CCC CCC TGG Pro Pro Trp joining region C 1 C L CD2 (5865) V L sheet GGG linker C between V L /V gives: CCC CGG Pro Arg near joining region 3. somatic mutation, 320 point mutations into the germline variable regnion to increase affinity. Germiline diversity of ~10 8 variable regions V region point mutations, affinity maturation M 1 CD1 (3036) 4. multiple constant regions Loop CD3 (95109) IgM IgD IgG IgE IgA Antibodies are expressed at the surface of preb cells. When an antigen binds it crosslinks two antibodies on the surface, sending signals to the B cell to proliferate & undergo somatic mutation.

2 Antibodies as catalysts yme have evolved maximal affinity towards T For more complex reactions: Ex. Cope rearrangement ow would you make an enzymelike catalyst for the reaction: sec + Make antibodies to: k cat /k uncat ~10 4 Want an immunogen like: ammond's postulate: for high energy intermediates, the structure of the transition state is similar to that of the intermediate P transition state analogue: tetrahedral, negativelycharged, stable Take albumin, covalently attach the ligand P k cat /k uncat =~

3 Ex. Aldol eaction: ' ' yme catalysis: 1. T stabilization 2. proximity effects 3. covalent catalysis 4. general acid base catalysis 5. cofactors pk a ~20 ymes make a chiffbase: 2 Make an immunogen: ' If any antibodies have a Lysine in the binding pocket: 4. General base catalysis Logk = Bronsted coefficient (pk a ) + C PLP catalyzed reactions: Lys Lys Asp ote pk a is ~67, Lys ~10 Lys C 2 2 C 2 k cat 3 C 2 C 2 C 2 LysE C 2 ' tautomerize ' table adduct 2 C k cat /k m ~10 9 k uncat

4 trategy mutate K258A inactive (with respect to + transfer) <10 6 of wt activity no large changes observed in crystal structure "rescue" activity with exogenous amines Use Lcysteine sulfinate as substrate (Asp analog with greater reactivity) 2 C C 3 + Asp yme Inhibitors: antibiotics Ex. ulfadrugs 2 2 reduced in vivo e / Prodrug chanism of action: paraaminosulfonamide esults linear dependence of rate versus [amine] plot of logk vs pk a was poor plot of log k vs "A 3 " Linear (/Et/Pr/Bu) with same pk a (~10.6) therefore use double linear plot: logk B = = (pk a ) + V + C V =( ) 3 c= % of full + charge on Lys 258 in T total variation: 10x greater due to volume than k cat due to basicity points to the effect of cavities/steric effects 2 2 F 3 C C dihydropteroate synthesis P P 2 dihydrofolate Bacteria utilize paraaminosulfonamide and substitute it into dihydrofolate synthesis, resulting in an inactive cofacter. 2 2

5 Ex. lactam penicillins Cell wall biosynthesis: DAla DAla AGAMAG LAla DyGlu L Lys (Gly) 5 2 Fast er (transpeptidase) L Lys (Gly) 5 2 er 2 (Gly) 5 Lys L cytoplasm C 2 cell wall biosynthesis enzymes porins L Lys (Gly) 5 2 (Gly) Lys 5 L inner membrane outer membrane mimic : C 2 peptidoglycan with: C 2 For resistance make a transpeptidase C 2 lactamase er C 2 er C 2 inactivated enzyme er C 2 slow deacylation C 2 2 yme catalyzed fast! C 2

GENETIC BASIS OF ANTIBODY STRUCTURE AND DIVERSITY. Steven J. Norris, Ph.D

GENETIC BASIS OF ANTIBODY STRUCTURE AND DIVERSITY Steven J. Norris, Ph.D Topics I. General principles II. The heavy chain Ig locus and VDJ rearrangement III. Light chain rearrangement. IV. Mechanisms of