Interactions Between the a and Subunits

The contact surface between the a and /3 subunits is very broad ( 1100 A2) and is mostly hydrophobic in character.7 The a subunit loops following strands 2,3,4, and 5 (Fig. 7.2) make important contacts with the /3 subunit. The 50-residue subdomain of the /3 subunit (residues 260-310) comprised of several long /3 hairpin loops makes several contacts with the a subunit (Fig. 7.5).7 Contacts in this region probably prevent tryptic cleavage of the /3 subunit in the 02/82 complex.34-35 The fact that the sites of tryptic cleavage of the /3 subunit (Lys-272, R-275, and K-283) are close to the a/(3 contact surface may explain why proteolytically cleaved /3 subunit does not associate with the a subunit.59-106 In contrast, cleavage of the /3 subunit by proteinase K at Glu-296, a residue at a position in the subdomain (residues 260-310) that does not make direct contact with the a subunit, yields an enzymatically active nicked f3 subunit that interacts weakly with a subunits.61  

Several single amino acid substitutions alter association between the a and /3 subunits. Two of the six conserved prolines in the a subunit (residues 57 and 132) are located close to or at the a//3 contact surface.107 Substitution of residues 57 and 132 by glycine or alanine 

The /8 subunit of tryptophan synthase from E. coli is inactivated by a mutation at Gly-281 (G281R).108 Gly-281 is located at a sharp turn in the trypsin-sensitive, subdomain (residues 260-310) that makes several contacts with the a subunit.7) The G281R mutation alters the catalytic properties of the isolated /8 subunit and weakens association with the a subunit. The mutation may interfere with hydrophobic interactions between the N-terminal and C-terminal domains of the /8 subunit and prevent a conformational change that affects catalytic properties and subunit interaction.108 Insertion of arginine or tryptophan between tyrosine 279 and phenylalanine 280 of the /3 subunit greatly weakens subunit interaction and decreases catalytic activity (X.-J. Yang and E.W. Miles, unpublished results). 

There are several types of evidence that the L-serine derivative that activates the a reaction is the Schiff base formed between aminoacrylate and pyridoxal phosphate (ES III in Fig. 7.6). (1) Amino acids including l- or D-tryptophan and glycine that form tetrahedral, 

Schiff base, or quinoidal intermediates do not appreciably affect the rate of the a reaction.90113 (2) L-serine and amino acids such as O-methyl-L-serine that form ES III do stimulate the a reaction.113 (3) The rate of indole-3-glycerol phosphate turnover is roughly correlated with the rate of formation of ES III for each of the amino acids.90-113 (4) The kinetics of the lag in cleavage of indole-3-glycerol phosphate and synthesis of L-tryptophan under single turnover conditions correspond to the rate of ES III formation 

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