Evidence for two conformers of the beta subunit of tryptophan synthase in solution

Ahmed, Salman; Miles, Edith Wilson

doi:10.1016/s0021-9258(18)50092-2

Cited by 5 publications

(3 citation statements)

References 41 publications

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“…This mechanism was first elucidated by Metzler et al (10,11) for the reactions of aspartate aminotransferase and glutamate decarboxylase with a quasisubstrate, L-serine-O-sulfate. We demonstrated this type of inactivation in the reactions of several mutant tryptophan synthase R 2 β 2 complexes and of the wild-type β 2 subunit with β-chloro-L-alanine (12,13). Inactivation results from displacement of aminoacrylate from the key E-AA intermediate followed by nucleophilic attack by the β-carbon of aminoacrylate on the internal aldimine which forms a covalent adduct (E-I, Scheme 1).…”

Section: Discussionmentioning

confidence: 96%

“…This conformation may be more active or may undergo inactivation less readily than the conformation that is stabilized by Na + . Different cations affect the extent of inactivation of the wild-type β 2 subunit by β-chloro-L-alanine (13). DL-R-Glycerol 3-phosphate may reduce the rate of formation of pyruvate and NH 3 by stabilizing the closed conformation that disfavors the β elimination reaction (12,34).…”

Section: Discussionmentioning

confidence: 99%

“…In this paper, we examine the effects of mutational alteration of Ser377 on the activities and spectroscopic properties of the β 2 subunit and R 2 β 2 complex. An important finding is that the S377D and S377E R 2 β 2 complexes undergo irreversible substrate-induced inactivation by a mechanism first elucidated by Metzler et al (10,11) and later found with different mutant forms of the tryptophan synthase R 2 β 2 complex and with the wild-type β 2 subunit (12,13). This inactivation results from displacement of aminoacrylate from the key E-AA intermediate followed by reaction of aminoacrylate with the methylene carbon of enzyme-bound PLP which forms a covalent adduct (E-I, Scheme 1).…”

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Tryptophan Synthase Mutations That Alter Cofactor Chemistry Lead to Mechanism-Based Inactivation

Jhee

McPhie

et al. 1998

Biochemistry

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Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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