ENZYMES A protein with catalytic properties due to its power of specific activation

Chemical reactions Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY During this part of the reaction the molecules are said to be in a transition state.

Reaction pathway

Making reactions go faster Increasing the temperature make molecules move faster Biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. They create a new reaction pathway “a short cut”

An enzyme controlled pathway Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.

Enzyme structure Enzymes are proteins They have a globular shape A complex 3-D structure Human pancreatic amylase © Dr. Anjuman Begum

The active site One part of an enzyme, the active site, is particularly important The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily © H.PELLETIER, M.R.SAWAYA ProNuC Database

Cofactors An additional non-protein molecule that is needed by some enzymes to help the reaction Tightly bound cofactors are called prosthetic groups Cofactors that are bound and released easily are called coenzymes Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors Jmol from a RCSB PDB file © 2007 Steve Cook H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

The substrate The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site

The Lock and Key Hypothesis Fit between the substrate and the active site of the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock. Temporary structure called the enzyme-substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate

The Lock and Key Hypothesis Enzyme may be used again Enzyme-substrate complex E S P Reaction coordinate

The Lock and Key Hypothesis This explains enzyme specificity This explains the loss of activity when enzymes denature

The Induced Fit Hypothesis Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html This explains the enzymes that can react with a range of substrates of similar types

Factors affecting Enzymes substrate concentration pH temperature inhibitors

Substrate concentration: Non-enzymatic reactions Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration

Substrate concentration: Enzymatic reactions Reaction velocity Substrate concentration Vmax Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme then Vmax will change too.

Initial reaction rate / arbitrary units Enzymes and [S] As soon as a reaction begins, [S] begins to fall and so it is important that initial reaction rates are measured Initial reaction rate / arbitrary units [S]

Initial reaction rate / arbitrary units Enzymes and [S] Initial reaction rate / arbitrary units [S]

Initial reaction rate / arbitrary units Enzymes and [S] Increasing [S] increases collision rate and increases reaction rate Initial reaction rate / arbitrary units [S]

Enzymes and [S] All active sites are not occupied All active sites are occupied. Enzymes are working at maximum rate. Initial reaction rate / arbitrary units All active sites are not occupied [S]

Enzymes and [Substrate] Maximum turnover number or Vmax has been reached Initial reaction rate / arbitrary units [S]

Enzymes and [enzyme] Can we explain this in terms of the proportions of active sites occupied? Initial reaction rate / arbitrary units What factor is limiting here? [Enzyme]

Enzymes and temperature: a tale of two effects Collision rate of enzymes and substrates Reaction rate / arbitrary units Number of enzymes remaining undenatured Temperature / oC

Enzymes and temperature Increasing kinetic energy increases successful collision rate Reaction rate / arbitrary units Temperature / oC

Enzymes and temperature Permanent disruption of tertiary structure leads to loss of active site shape, loss of binding efficiency and activity Reaction rate / arbitrary units Temperature / oC

Enzymes and temperature Optimum temperature Reaction rate / arbitrary units Temperature / oC

Enzymes and pH The precise shape of an enzyme (and hence its active site) depends on the tertiary structure of the protein Tertiary structure is held together by weak bonds (including hydrogen bonds) between R-groups (or ‘side-chains’) Changing pH can cause these side chains to ionise resulting in the loss of H-bonding…

Enzymes and pH Optimum pH Either side of the optimum pH, the gradual ionising of the side-chains (R-groups) results in loss of H-bonding, 3o structure, active site shape loss of binding efficiency and eventually enzyme activity Reaction rate / arbitrary units pH

Enzymes and pH Optimum pH This loss of activity is only truly denaturation at extreme pH since between optimum and these extremes, the loss of activity is reversible Reaction rate / arbitrary units pH

Enzymes and pH

Enzymes and inhibitors Inhibitors are molecules that prevent enzymes reaching their maximum turnover numbers Some inhibitors compete with the substrate for the active site Some inhibitors affect the active site shape by binding to the enzyme elsewhere on the enzyme Active site directed inhibition Non-active site directed inhibition

Active site directed inhibition (Competitive) Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate Inhibitor Enzyme

Active site directed inhibition (Competitive) Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate Enzyme activity is lost Enzyme/Inhibitor complex

Enzymes and competitive inhibition At low [S], the enzyme is more likely to bind to the inhibitor and so activity is markedly reduced Initial reaction rate / arbitrary units Uninhibited Inhibited [S]

Enzymes and competitive inhibition As [S] rises, the enzyme is increasingly likely to bind to the substrate and so activity increases Initial reaction rate / arbitrary units Uninhibited Inhibited [S]

Enzymes and competitive inhibition At high [S], the enzyme is very unlikely to bind to the inhibitor and so maximum turnover is achieved Initial reaction rate / arbitrary units Uninhibited Inhibited [S]

Non-active site directed inhibition (Non-competitive) Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Inhibitor Enzyme

Active site is changed irreversibility Non-competitive inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Active site is changed irreversibility Enzyme

Non-competitive inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Activity is permanently lost Enzyme

Enzymes and non-competitive inhibition Can we explain this graph in terms of limiting factors in the parts of the graph A and B? Initial reaction rate / arbitrary units [S] A B

Applications of inhibitors Negative feedback: end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases. Medicine antibiotics, sulphonamides, sedatives and stimulants

The "kinetic activator constant" Understanding Km The "kinetic activator constant" Km is a constant Km is a constant derived from rate constants Km is an approximation of the dissociation constant of E from S Small Km means tight binding; high Km means weak binding

The theoretical maximal velocity Understanding Vmax The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is increased

A measure of catalytic activity The turnover number A measure of catalytic activity kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. Values of kcat range from less than 1/sec to many millions per sec

Michaelis-Menten equation

Lineweaver-Burke plot

Hanes-Woolf plot