Involvement of α-Cysteine-62 and β-Tryptophan-135 in Human Pyruvate Dehydrogenase Catalysis

• Published in: Archives of biochemistry and biophysics 1999-09, Vol.369 (2), p.277-287
• Main Authors: Korotchkina, Lioubov G., Showkat Ali, M., Patel, Mulchand S.
• Format: Article
• Language: English
• Subjects: bromopyruvate; Catalytic Domain; Cysteine - genetics; Humans; Kinetics; Mutagenesis, Site-Directed; pyruvate dehydrogenase; Pyruvate Dehydrogenase (Lipoamide); Pyruvate Dehydrogenase Complex - antagonists & inhibitors; Pyruvate Dehydrogenase Complex - genetics; Pyruvate Dehydrogenase Complex - metabolism; Pyruvates - pharmacology; Pyruvic Acid - analogs & derivatives; Pyruvic Acid - pharmacology; substrate analogs, fluoropyruvate; thiamin pyrophosphate; Tryptophan - genetics
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1364

Pyruvate dehydrogenase (E1), a heterotetramer (α2β2), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1α (αC62) and tryptophan-135 of E1β (βW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: αC62A and βW135L. Compared to wild-type, kcat values for αC62A and βW135L measured by PDC assay were markedly reduced to 7.2 and 11.6%, respectively. Apparent Km 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. Km values for pyruvate increased only moderately (twofold). The αC62A and βW135L 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 αC62A and βW135L 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 αC62 and βW135 facilitate coenzyme binding, and αC62 could be near the substrate-binding site.