Enzymology Part 2 - Enzyme Kinetics lec

Competitive inhibition: Example 2, ______________ dehydrogenase

Alcohol dehydrogenase ngested methanol is converted to formaldehyde and formic acid by alcohol dehydrogenase (ADH).

Lineweaver-Burk double reciprocal plot showing the effects of a non-competitive inhibitor. Vmax _____________ (is reduced for an inhibitor. Make sure you recall that in a double reciprocal plot, a higher value on the y-axis means a lower Vmax). Km is_______________. Lineweaver-Burk double reciprocal plot showing the effects of a non-competitive inhibitor.

Changes Unchanged

_______________ Inhibitors bind the active site and block activity.

Competitive

Statins are competitive inhibitors of ____________ reductase.

HMG-CoA EX: Atorvastatin Lipitor Simvastatin Zocor Pravastatin Pravachol Rusuvastatin Crestor Fluvastatin Lescol Lovastatin Mevacor

Hexokinase comes in two isozyme forms: Both catalyze Glu ---> Glu-6-P, the initial step in Glycolysis.

Hexokinase: muscle and most other tissues Glucokinase: Liver and pancreas.

What is the meaning of Km and Vmax

Km is the most important parameter - Is robust from assay to assay - Is independent of enzyme concentration - Indicates concentration range for activity Vmax is particular to the assay - Depends on the amount of enzyme - Depends on the kcat - kcat is better for comparing enzymes

_______________ inhibitor binds to a regulatory site (not the active site) and modifies the rate of reaction.

Non-competitive

__________________Occurs by binding of an inhibitor, I, outside the active site. The binding of I does not interfere directly with substrate The binding of I likely influences the conformation of the enzyme to reduce (or enhance) activity. The principal change is in Vmax.

Noncompetitive Inhibition

The Km reflects both the binding of the substrate and the rate of catalysis: • weak binding (low affinity), implies higher Km.

Note: Binding constant = KD = k-1/k1, while Km = (k2 + k-1)/k1; they are not identical.

_____________ Inhibitors This reduces Vmax • No effect on Km React Covalently with the enzyme • Effectively reduces the concentration of enzyme

Suicide inhibators Aspirin (acetylsalicylate). Inactivates enzymes such as cyclooxygenase (COX). COX is a critical enzyme for making prostaglandins from the fatty acid arachidonic acid in the inflammatory response. Nerve gases (DFP, Sarin, Echothiphate, etc.) irreversible inhibitors of acetylcholine esterase (the enzyme that inactivates acetylcholine) Esterases work by the same general mechanism and design as proteases. The serine here that normally catalyzes ester cleavage works in the same manner as the catalytic serine in trypsin. Nerve gases are very potent and deadly compounds. Proton Pump Inhibitors (PPIs). Drugs that decrease acid secretion in stomach. Penicillin antibiotics - Penicillin type antibiotics work by inhibiting the enzymes that synthesize bacterial cell walls. The enzymes have a structure also like proteases, with an active site serine. In this case, the antibiotics are really very slow cleavable substrates. But this effectively makes them suicide inhibitors.

_____________ inhibitors Behave like a substrate, but become covalently attached to the enzyme.

Suicide inhibitors

__________________ Inhibitor binds only in the presence of substrate and blocks activity.

Un-competitive

in ______________________ An inhibitor binds only to the Enzyme-substrate complex (ES). results in changes in both Vmax and Km.

Un-competitive inhibition is very rare; one relevant example: Lithium inhibition of inositol monophosphatase

____________________ inhibition Intercepts change: Both x and y • Vmax changes (decreases) • Km changes (decreases) • Vmax/Km is constant - Vmax and Km change the same fold.

Un-competitive inhibition: Lineweaver-Burk Plot

Graphs of enzymes

Various types of inhibition shown as MM plots and as double reciprocal plots • A - no inhibitor • B - Competitive Inhibition • C - Noncompetitive inhibition or suicide inhibitor • D - Uncompetitive inhibition

Competitive Inhibition Effects of a competitive inhibitor are readily seen as changes in a Lw-B plot Vmax is unchanged Km is increased

Vmax= unchanged Km= increased Overall: this is a slope effect. You can see that in the equation - only the slope is affected, not the y-intercept.

The regulatory sites can activate (ADP) or inhibit (G6P) enzyme activity. Allosteric regulation acts at sites different from the active site to increase or decrease activity through dynamic structural changes in the protein. Allosteric inhibitors exhibit __________________ inhibition.

noncompetitive

Michaelis-Menten equation

v= vmax * (([S] / (Km + [S])))