Involvement of alpha-cysteine-62 and beta-tryptophan-135 in human pyruvatedehydrogenase catalysis

Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the fir st catalytic component of the mammalian pyruvate dehydrogenase complex (PDC ), To investigate the roles of cysteine-62 of E1 alpha (alpha C62) and tryp tophan-135 of E1 beta (beta W135) (identified previously as active site res idues using chemical modifications) in El catalysis, two recombinant human El mutants were generated using site-directed mutagenesis: alpha C62A and b eta W135L, Compared to wild-type, k(cat) values for alpha C62A and beta W13 5L measured by PDC assay were markedly reduced to 7.2 and 11.6%, respective ly. Apparent K-m values for thiamin pyrophosphate (TPP) were increased appr oximately sixfold for both mutants, resulting in catalytic efficiency for T PP of only 1-2% of the wild-type E1. K-m values for pyruvate increased only moderately (twofold), The alpha C62A and beta W135L mutants were less ther mostable than wildtype El. The conformations of the mutant apo-Els determin ed by spectral analysis were different from that of the wild-type apo-E1, C D spectral analysis indicated that TPP binding was affected for both the al pha C62A and beta W135L mutant Els, The substrate analogs, fluoropyruvate a nd bromopyruvate, were shown to be active site-directed inhibitors of human El; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibite d human E1 due to SH-group modification, Pyruvate induced inactivation of h uman EI could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in El inhibition by the substrate and su bstrate analogs, Taken together these results indicate that alpha C62 and b eta W135 facilitate coenzyme binding, and alpha C62 could be near the subst rate-binding site. (C) 1999 Academic Press.