Evidence for a Rate-determining Proton Abstraction in the Serine Deaminase Reaction of the β2 Subunit of Tryptophan Synthetase

The β2 subunit of tryptophan synthetase of Escherichia coli catalyzes the pyridoxal-P-dependent conversion of l-serine to pyruvate and ammonia. In order to study the effect of substitution of deuterium for the α-hydrogen of serine on the rate of this reaction, a new method for the synthesis of α-deutero-dl-serine has been developed; analyses of the product by mass spectroscopy and nuclear magnetic resonance spectroscopy are shown. The rate of pyruvate formation from α-deuteroserine is one-fourth of that from α-proteoserine in the absence of NH4+ ion. This kinetic isotope effect indicates that the dissociation of the α-C—H bond of l-serine is the rate-determining step in the formation of pyruvate under these conditions. The enzyme-substrate intermediate which accumulates before the rate-determining α-proton abstraction under these conditions can be observed by its absorption at 420 nm or by its fluorescence. We conclude that this intermediate must be the Schiff base formed between pyridoxal-P and l-serine. The disappearance of this intermediate formed from α-deutero-dl-serine and α-proteo-dl-serine has been measured by stopped flow experiments in the presence of NH4+ ion which increases the rate of this step. The results show a kinetic isotope effect of 4.6 on the rate constant for the forward reaction in which this intermediate disappears. These results are direct evidence that a proton is transferred in this step.