Hydrogen exchange kinetics and the mechanism of reaction B of yeast tryptophan synthase

Abstract

It has been shown that yeast tryptophan synthase( l-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20) catalyses tritium exchange reactions between protons on the α-carbon of l-serine or l-tryptophan, and water. The absolute rates of these reactions and indole-serine condensation (reaction B), all of which are pyridoxal phosphate-dependent, were measured. l-Serine exchange was resolved into two components, a high-affinity, slow, Michaelian reaction ( K m,H s = 0.06 mM, k cat,H s 3·10 −3 s −1) and a faster reaction ( k cat > 2.5 s −1) which was not saturated even at 100 mM l-serine. Hydrogen exchange by tryptophan was a Michaelian process ( K m,H T = 2.9 mM; k cat,H T = 0.6 s −). Indole did not inhibit either exchange reaction. A plausible explanation of the results, that reaction B has a ping-pong mechanism with serine as first substrate and water and l-tryptophan as first and second products, respectively, was inadequate because of the observations that l-tryptophan is synthesised with less than 1 mol of exchanged proton per mol amino acid, and that the ratio k cat/ K m for serine changes between enzyme reactions. A branched modification with two enzyme-serine complexes, only one of which will exchange protons with water, will fit all the results.