L-Serine binds to arginine-148 of the .beta.2 subunit of Escherichia coli tryptophan synthase

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Abstract
Inactivation of the .beta.2 subunit and of the .alpha.2.beta.2 complex of tryptophan synthase of E. coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of 1 arginyl residue/.beta. monomers. The substrate L-serine protects the holo .beta.2 subunit and the holo .alpha.2.beta.2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by the holo .beta.2 subunit and the holo .alpha.2.beta.2 complex indicate that L-serine and phenylglyoxal bind to the same essential arginyl residue in the holo .beta.2 subunit. The apo .beta.2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo .beta.2 subunit binds pyridoxal phosphate and the .alpha. subunit but cannot bind L-serine or L-tryptophan. The .alpha.-carboxyl group of L-serine and to the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the .beta.2 subunit. The specific arginyl rsidue in the .beta.2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal was identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains 3 other basic residues (lysine-137, arginine-141 and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148 and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium and yeast supports a functional role for these 3 residues in anion binding. The location and role of the active-site arginyl residues in the .beta.2 subunit and in 2 other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.