Factors Affecting Enzyme Activity Enzymes are large globular proteins They have a precise 3-D shape Some have quaternary structure The active site (blue) represents a tiny part of the molecule RuBisCo Amino Acid A reminder about protein structure Amylase Protein structure is achieved by the precise folding of secondary structures to form a tertiary structure held together by a range of bond types between R- groups (or side-chains) Some reaction kinetics The Lock and Key analogy Induced fit Enzymes and temperature: a tale of two effects Temperature / o C Collision rate of enzymes and substrates Number of enzymes remaining undenatured Reaction rate / arbitrary units Enzymes and temperature Temperature / o C Increasing kinetic energy increases successful collision rate Reaction rate / arbitrary units Enzymes and temperature Temperature / o C Permanent disruption of tertiary structure leads to loss of active site shape, loss of binding efficiency and activity Reaction rate / arbitrary units Enzymes and temperature Temperature / o C Optimum temperature Reaction rate / arbitrary units 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 pH Reaction rate / arbitrary units Either side of the optimum pH, the gradual ionising of the side-chains (R-groups) results in loss of H- bonding, 3 o structure, active site shape loss of binding efficiency and eventually enzyme activity Optimum pH Enzymes and pH pH Reaction rate / arbitrary units This loss of activity is only truly denaturation at extreme pH since between optimum and these extremes, the loss of activity is reversible Optimum pH Enzymes and pH Enzymes and [S] [S] Initial reaction rate / arbitrary units As soon as a reaction begins, [S] begins to fall and so it is important that initial reaction rates are measured Enzymes and [S] [S] Initial reaction rate / arbitrary units Enzymes and [S] [S] Initial reaction rate / arbitrary units Increasing [S] increases collision rate and increases reaction rate Enzymes and [S] [S] Initial reaction rate / arbitrary units All active sites are occupied. Enzymes are working at maximum rate. All active sites are not occupied Enzymes and [S] [S] Initial reaction rate / arbitrary units Maximum turnover number or V max has been reached Enzymes and [enzyme] [Enzyme] Initial reaction rate / arbitrary units Can we explain this in terms of the proportions of active sites occupied? What factor is limiting here? 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 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 Inhibitor resembles the substrate enough to bind to active site and so prevent the binding of the substrate: Substrate Enzyme/Inhibitor complex Enzyme activity is lost Enzymes and active site directed inhibition [S] Initial reaction rate / arbitrary units At low [S], the enzyme is more likely to bind to the inhibitor and so activity is markedly reduced Uninhibited Inhibited Enzymes and active site directed inhibition [S] Initial reaction rate / arbitrary units As [S] rises, the enzyme is increasingly likely to bind to the substrate and so activity increases Uninhibited Inhibited Enzymes and active site directed inhibition [S] Initial reaction rate / arbitrary units At high [S], the enzyme is very unlikely to bind to the inhibitor and so maximum turnover is achieved Uninhibited Inhibited Non-active site directed inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Inhibitor Enzyme Non-active site directed inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Enzyme Active site is changed irreversibility Non-active site directed inhibition Inhibitor does not resemble the substrate and binds to the enzyme disrupting the active site Substrate Enzyme Activity is permanently lost Enzymes and non-active site directed inhibition [S] Initial reaction rate / arbitrary units Can we explain this graph in terms of limiting factors in the parts of the graph A and B? AB