Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase

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Abstract
Clostridial [Clostridium kluyveri] phosphotransacetylase [EC 2.3.1.8] catalyzes acyl group transfer between CoA and Pi and also the arsenolysis of acetyl-CoA (AcCoA) to yield acetate and CoA-SH. The enzyme mobility on sodium dodecyl sulfate electrophoresis corresponds to a MW of 70,000. Kinetics of forward and reverse reactions are of the ternary type as previously reported and product inhibition data are consistent with a random binding scheme. One essential sulfhydryl group per 70,000 daltons was inactivated in a pseudo-1st-order process by N-ethylmaleimide or 5,5''-dithiobis(nitrobenzoic acid). Reduction of this inactivation rate by 50% in the presence of AcCoA or acetyl phosphate concentrations near their kinetic .hivin.K values demonstrates binding of these acyl donors in simple enzyme-substrate complexes. Pulse-chase experiments show that these binary complexes are functional and that they do not dissociate rapidly compared with their catalytic turnover rates. Incubation of the enzyme with 14C-labeled acyl donors failed to produce labeled protein after passage through Sephadex. This was true despite efforts to mimic substrate synergism with desulfo-CoA or to compensate for unfavorable equilibria by CoA traps. Very slow isotope exchange reactions of 32Pi into acetyl phosphate and [3H]CoA into AcCoA were at first observed. These were shown to be artifacts of contamination by 2nd substrates. Attempts to detect exchange reactions between acetyl phosphate and Pi, even in the presence of the CoA analogue, desulfo-CoA, were also unsuccessful. Therefore, no evidence for an acyl-enzyme was detected.

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