The Steady State Assumption BMED282 U2L3 Enzyme s and Regulation [conc] Product Substrate Time (min) The Steady State Assumption Prac Results: Alkaline Phosphatase [conc] Total Enzyme added = [] + [Efree] 2 Michaelis-Menten Time (min) [] [Enzyme free ] Velocity (nmol/min) 2 1 1. 1 1. 2 [S] (mm).12.8.6.4.2. Prac Results: Alkaline Phosphatase Lineweaver-Burke y =.9x +.431 R 2 =.9981.. 1. v Velocity (nmol/min) Michaelis-Menten 2 2 1 1. 1 1. 2 [S] (mm) Eadie-Hofstee 2 y = -.138x + 23.38 2 R 2 =.999 1 1 2 4 6 8 1 12 v/[s] [S]/v Lineweaver-Burke.12.8.6.4 y =.9x +.424.2 R 2 =.998... 1. Hanes-Woolfe y =.429x +.6.9 R 2 =.9998.8.7.6..4.3.2.1.. 1 1. 2 [S] 1
The Michaelis Menten Relationship By lots of clever maths, which we won t go over at 3:3 on a Wednesday afternoon The equation below describes the hyperbola. Velocity = * [S] ([S] + Km) This measurement reflects the efficiency of the enzyme. What we really want to know is how often goes to. An efficient enzyme will send all the to. An inefficient enzyme will send some back to E + S The ideal efficient enzyme is where every time E collides with S, product P is formed. The upper limit will be the rate of diffusion of the substrate and enzyme in the solution. This is ~1 8 to 1 9 (M) -1 sec -1. If the approaches 1 8 to 1 9 (M) -1 sec -1 the enzyme is considered perfect! K cat is k 2 So = k 2 * k 1 / (k -1 + k 2 ) Now if the enzyme is really efficient k -1 will be really small (very little going back to E + S) approaches k 1. This is limited by the rate of diffusion. s s can be irreversible or reversible. Irreversible inhibitors usually covalently bind to the enzyme; they are often slower to act (time dependent inhibition) and present as non-competitive inhibition. They cannot be dialysed out or diluted out. Reversible inhibitors; competitive, noncompetitive, mixed, uncompetitive The Competitive The inhibitor binds to the same site on the enzyme as the substrate Thus it competes with the substrate It eventually reaches It needs more substrate to do it Km increases 2
Competitive Inhibition The Effect of a Competitive Reaction rate The competitive Is in yellow K M [substrate] The Competitive The Competitive A Competitive A Competitive unchanged Km increases -1/Km increases No No The Non-Competitive Non-competitive Inhibition The inhibitor binds to a site other than the substrate binding site Both I and S can bind to the enzyme simultaneously It never reaches The Km does not change It is like adding less enzyme to an assay S K' I 3
The Effect of a Non-competitive The Non-competitive : A Non-competitive Reaction rate The non-competitive Is in yellow No K M [substrate] The Non-competitive : A Non-competitive No decreases increases Km unchanged The mixed Non-Competitive The inhibitor binds to a site other than the substrate binding site But the binding of the inhibitor affects the binding of the inhibitor Both Km and change The most common type of inhibition Non-competitive Inhibition The mixed non-competitive : A mixed non-competitive K' I No S K i K i 4
The Uncompetitive The inhibitor binds to the complex only Both Km and decrease /Km unchanged Uncompetitive Inhibition S The Uncompetitive : Regulatory Enzymes An uncompetitive No decreases Km decreases /Km unchanged Allosteric regulation: the binding of a small molecule (ligand) distant from the active site, Covalent modification, often phosphorylation of serine, threonine or tyrosine residues, Regulatory Enzymes Changes in the amount of enzyme either by changes in gene expression, (often at the level of transcription) or protein turnover, Substrate availability, Inhibition (competitive etc), Activation of zymogens or proenzymes.