Involvement of alpha-cysteine-62 and beta-tryptophan-135 in human pyruvate dehydrogenase catalysis.
Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1alpha (alphaC62) and tryptophan-135 of E1beta (betaW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: alphaC62A and betaW135L. Compared to wild-type, k(cat) values for alphaC62A and betaW135L measured by PDC assay were markedly reduced to 7.2 and 11. 6%, respectively. Apparent K(m) values for thiamin pyrophosphate (TPP) were increased approximately sixfold for both mutants, resulting in catalytic efficiency for TPP of only 1-2% of the wild-type E1. K(m) values for pyruvate increased only moderately (twofold). The alphaC62A and betaW135L mutants were less thermostable than wild-type E1. The conformations of the mutant apo-E1s determined by spectral analysis were different from that of the wild-type apo-E1. CD spectral analysis indicated that TPP binding was affected for both the alphaC62A and betaW135L mutant E1s. The substrate analogs, fluoropyruvate and bromopyruvate, were shown to be active site-directed inhibitors of human E1; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibited human E1 due to SH-group modification. Pyruvate induced inactivation of human E1 could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in E1 inhibition by the substrate and substrate analogs. Taken together these results indicate that alphaC62 and betaW135 facilitate coenzyme binding, and alphaC62 could be near the substrate-binding site.
Korotchkina
L G
LG
School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, 14214, USA.
Showkat Ali
M
M
Patel
M S
MS
D020134:Catalytic Domain; D003545:Cysteine / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D019289:Pyruvic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D014364:Tryptophan / Q000235:genetics
D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; C017319:3-fluoropyruvate; C017092:bromopyruvate; D019289:Pyruvic Acid; D014364:Tryptophan; D030481:Pyruvate Dehydrogenase (Lipoamide); D003545:Cysteine