Evidence for a rate-determining proton abstraction in the serine deaminase reaction of the beta 2 subunit of tryptophan synthetase.

ion of the a-proton. The rate constant for the converkz sion of Intermediate 1 to Intermediate II with [a-Hlserine as Therefore, /L.~ = kZ = 3.17 s-1. substrate which we have obtained from stopped flow absorption For [a-Dlserine, kobs = 2.74 s-l = kz + kmz; kLJkz = 3. measurements (kobs = 6.3 s-l) is about 2 times higher than the Therefore, k-2 = 3 kt; k-z = 2.05 s-1; and kp = 0.685 s-1. rate constant obtained by York (kobs = 3.6 s-1) using stopped Deuterium isotope effects can be calculated for each rate conflow fluorescence measurements at a lower protein concentration stant from these data: (9). These numbers are in reasonably close agreement and provide further evidence that the fluorescence and absorbance obhi served under these conditions are due to the same intermediate. = 4.6 for kz and 2 = 1.5 for kwz. ko Two enzyme-substrate intermediates accumulate in the presence of NH4+ ion under steady state conditions: Intermediate 1 and an intermediate which absorbs at 330 nm. York has shown that the latter intermediate, which he terms the “pale” species, is formed as one-half of Intermeclia,te 1 disappears before the steady state condition is reached (9). Therefore, the reaction of the ps subunit with L-serine in the presence of NHd+ ion appears to contain two rate-limiting steps which occur after each of the two enzyme-substrate intermediates which accumulate under steady state conditions (9). These observations can be represented by a minimal kinetic scheme (Equation 1) in which ES-I represents Intermediate 1 which absorbs at 420 nm and ES-II represents the “pale” species which absorbs at 330 nm. One or more other enzyme-substrate intermediates connected by fast steps could occur between ES-I and ES-II without affecting t,his kinetic scheme. TTS k E-tS & ES-I < k, / , k, k-&+ ES-II k-1 E + P (1)