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