Transient Kinetic Analysis of the Interaction of l-Serine with Escherichia coli d-3-Phosphoglycerate Dehydrogenase Reveals the Mechanism of V-Type Regulation and the Order of Effector Binding

摘要

Pre-steady state stopped-flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenasen(PGDH) reveals that the physiological inhibitor, L-serine, exerts its effect on at least two steps in the kineticnmechanism, but to very different degrees. First, there is a small but significant effect on the dissociationnconstant of NADH, the first substrate to bind in the ordered mechanism. The effect of serine is mainly on thenbinding off rate, increasing the Kd to 5 and 23 μMfrom 0.6 and 9 μM, respectively, for the two sets of sites innthe enzyme.Amore profound effect is seen after the second substrate is added. Serine reduces the amplitude ofnthe signal without a significant effect on the observed rate constants for binding. The serine concentration thatnreduces the amplitude by 50% is equal to the K0.5 for serine inhibition. The data are consistent with thenconclusion that serine binding eliminates a conformational change subsequent to substrate binding bynformation of a dead-end quaternary complex consisting of enzyme, coenzyme, substrate, and effector. Thus,nthe mechanistic basis for V-type regulation in this enzyme is a reduction in the population of active speciesnrather than a differential decrease in the velocity of active species. Pre-steady state analysis of binding of serinento a mutant PGDH (W139F/E360W) demonstrates that each serine binding interface produces an integratednfluorescent signal. The observed rate data are complex but conform to a model in which serine can bind to twonforms of the enzyme with different affinities. The integrated signal from each interface allows the amplitudendata to clearly define the order of binding to each site, and modeling the amplitude data with speciesndistribution equations clearly demonstrates an alternate interface binding mechanism and the direction ofnbinding cooperativity.