南京大学-杨荣武生物化学-英文版-第二篇-EnymeVII

1、Allostery & Cooperativity:,Kinetic Consequences & Structural Basis,Allostery: Websters Definition,Main Entry: allosteric Pronunciation: a-lO-ster-ik, -stir- Function: adjective Etymology: all- + steric Date: 1962 : of, relating to, or being a change in the shape and activity of a protein (as an enzy

2、me) that results from combination with another substance at a point other than the chemically active site - allosterically /-i-k(&-)lE/ adverb - allostery /a-lO-ster-E, -stir-/ noun,Allostery: Key Point,Binding of a ligand at a site different from the active site modulates the activity. This behavio

3、r extends well beyond the normal use of the word “allostery” which is often used to discuss cooperative interactions. The molecular basis for allostery provides insight into many regulatory mechanisms. That which has been learned by studying allosterically regulated enzymes/proteins has profoundly i

4、nfluenced our understanding of cooperativity and enzyme regulation in general.,Allostery vs Cooperativity,The terms allostery and cooperativity have been muddled. Allostery strictly refers to influence of activity by a distant site. Cooperativity indicates that the occupancy of one site in a multisu

5、bunit enzyme influences the binding on the others. This is a form of allostery, but is only one manifestation of a general phenomena. Unfortunately allostery had become almost exclusively associated with the behavior of multi-subunit enzymes.,Kinetic Signature of Cooperativity in Enzymes,Multisubuni

6、t enzymes that exhibit cooperativity show a sigmoidal initial velocity curve in contrast to the hyperbolic curve for independent subunits.,Kinetic Consequences of Allosteric Effectors on Cooperative Enzymes,This is the traditional view of feed-back inhibition and regulation in “allosteric” enzymes.,

7、Classic Examples of Allostery,Hemoglobin (not an enzyme) This was the origin of the T and R states Aspartate transcarbamoylase. Example of feedback inhibition These are great examples, but there are many others.,Types of Regulation,Homotrophic responses: This refers to allosteric modulation of enzym

8、e activity by substrate molecules. This necessarily must occur in multisubunit enzymes. Heterotrophic responses: This refers to regulation by non-substrate molecules or combinations of non-substrate and substrate molecules. Allosteric regulation can be positive or negative.,Models for Allosteric Reg

9、ulation,Concerted (conceptually simple and often effective) Sequential (probably correct but difficult to prove),Postulates of the Concerted Model,Allosteric enzymes are composed of identical protomers that occupy equivalent positions in the enzyme. Each protomer contains a binding site for each spe

10、cific ligand. Each protomer can exist in only one of two states. The R (relaxed or high substrate affinity state) or T (taut or low substrate affinity state). All protomers in enzyme molecule must be in either the R or T state. The R and T states are in equilibrium with each other. The binding affin

11、ity of a specific ligand depends on the conformation of the enzyme (R or T) and not on the neighboring site occupancy.,Hemoglobin,The concerted mechanism for allosteric cooperative enzymes arose from consideration of the binding curves of oxygen binding to hemoglobin. This was modeled as a two state

12、 transition from deoxy (T) to oxy (R) hemoglobin.,Deoxy (T State),Oxy (R State),Concerted Model,Assumes 2 conformation states: R & T Binding of substrate induces all subunits to change to R state. No T-R hybrids. Allows for + cooperativity only.,+ cooperativity,T state,R state,Simple Version of the

13、Concerted Model,This approximate model implies that the substrate does not bind to the inactive state. This must be a gross simplification but it explains the principle. Interestingly, it accounts for a lot of enzymatic behavior (it is the simplest model). It cannot explain negative cooperativity.,S

14、equential Model,The binding of substrate switches conformation of only the subunit to which it is bound. Conformational change in one subunit may or the affinity of other subunits have for the substrate. Allows for + or - cooperativity.,Both the sequential and concerted models account for allosteric

15、 regulation of cooperative enzymes,Allosteric activators that alter the Km of an enzyme stabilize the high affinity state of the active site. Allosteric activators that alter the kcat of an enzyme stabilize the high activity state of the active site.,Allosteric inhibitors that alter the Km of an enz

16、yme stabilize the low affinity state of the active site. Allosteric inhibitors that alter the kcat of an enzyme stabilize the low activity state of the active site.,Sequential Model,T,T,T,T,T,T,T,T,T,T,R,R,R,R,R,R,R,R,R,R,Sequential Model for Allosteric Regulation of Cooperative Enzymes,Sequential M

17、odel This too is a gross simplification of reality, but embodies the concept that each binding event generates a different (group) of conformational states. k1 etc are microscopic rate constants.,Simplified Rate Equations for the Sequential Model for a Highly Cooperative enzyme,This assumes that the

18、 concentrations of ES, ES2 .and Esn-1 are very small due to the high cooperativity. This is similar in form to the Hill equation.,n is referred to as the Hill constant and is a measure of the cooperativity of the enzyme. the greater the value of n, the higher the cooperativity. For the case where n

19、= 1 (no cooperativity)At 1/2Vmax S0.5 gives k = S0.5n. These parameters can be derived by non-linear regression analysis. This is the simplest kinetic model for cooperative enzymes.,Hill Plot,The velocity data for cooperative enzymes can be presented in a linear form by use of Equation :,The slope o

20、f the best fit line provides an estimated of the Hill coefficient n, and the y intercept provides an estimate of log(K).,Concerted vs Sequential,The concerted model contains the simplest assumptions and directly provides a kinetic estimate of the role of effectors (but it cannot explain negative cooperativity). Fundamentally, he sequential model must be correct, but embodies many more parameter