Abstract
Pyruvate formate-lyase has been demonstrated in Clostridium kluyveri, C. butylicum and C. butyricum. The properties of the clostridial enzyme have been studied in more detail in cell-free lysates of C. butyricum. The following activities were investigated: pyruvate cleavage to acetylBiochemisches Institut der Universitat BRD-7800 Freiburg i. Br., Hermann Herder-Strase 7 German Federal RepublicBiochemisches Institut der Universitat BRD-7800 Freiburg i. Br., Hermann Herder-Strase 7 German Federal RepublicCoA and formate, pyruvate synthesis from acetyl-CoA and formate, and formate exchange with the carboxyl group of pyruvate. Formate formation from pyruvate (substrate concentration for half-maximal velocity, ([S]0.5v= 1.6 mM) was dependent on coenzyme A ([S]0.5v= 0.012 mM). Pyruvate (100%) could be replaced to some degree by 2-oxobutyrate (31%), coenzyme A (100%) by dephosphocoenzyme A (40%). In contrast to all other known pyruvate formate-lyase reactions the clostridial reaction was readily reversible. Pyruvate synthesis from acetyl CoA ([S]0.5v= 0.19 mM) and formate ([S]0.5v= 10 mM) proceeded with 40% of the velocity of the forward reaction. The exchange reaction between pyruvate ([S]0.5v= 0.6 mM) and formate ([S]0.5v= 20 mM) was independent of CoA or phosphate. This activity was even inhibited in the presence of CoA. DEAE-cellulose-treated extracts did not catalyze formate formation from pyruvate. The activity could however be restored by reduced ferredoxin, Co2+· thiol or Fe2+· dithiol complexes. When the lysates were treated with Dowex-potassium or charcoal in addition to DEAE-cellulose, reactivation required both one of the electron donors and S-adenosylmethionine. Methionine, ATP, ADP, AMP, methionine plus ATP, or S-adenosylhomocysteine could not substitute for S-adenosylmethionine. Rather, S-adenosylhomocysteine antagonized the reactivation dependent on S-adenosylmethionine. A similar inactivation-activation behavior was observed for the pyruvate formation and the exchange activities. The pattern of enzymes related to the synthesis of C1 units by several clostridia demonstrates that either a pyruvate-formate-lyase or a CO2-reductase reaction leads to the formation of formate, which is required for the synthesis of C1 units in these bacteria.
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