Pyruvate Formate‐Lyase Reaction in Escherichia coli

Abstract

Fractionation of the cell extract led to partial isolation of four protein fractions that are involved in the dissimilation of pyruvate into acetyl-CoA and formate: Enzyme I , molecular weight about 140000 ; enzyme 11, molecular weight about 30000, which requires an activation by ferrous ion and dithiols; enzyme 111, molecular weight 25000, which is a flavoprotein with flavin-mononucleotide as coenzyme ; and a hitherto less characterized fraction IV. The system in addition comprises the cofactors S-adenosylmethionine and thiamine diphosphate.Enzyme 111, fraction IV, and thiamine diphosphate could be replaced by the reagents cobaltous ion and thiols which form complexes of high reducing power. This part of the enzyme system obviously plays an auxiliary role, most likely by providing an appropriate redox potential via pyruvate as electron donor.The ability to dissimilate pyruvate was found to arise only and specifically from an interaction between enzyme I, ferrous ion-activated enzyme I1 and adenosylmethionine, in presence of either of t,he above auxiliary systems. Enzyme I is thereby converted into an active form, possibly by reduction, which alone is responsible for the catalysis of the pyruvate dissimilation reaction. The identification of this pyruvate formate-lyase was accomplished by sedimentation experiments in anaerobic sucrose gradients. As a substitute for the classical "clastic reaction", the semi-systematic term "pyruvate formate-lyase reaction", which will be used here, has recently been introduced to designate reaction (1) [5].I n sharp contrast to the well established mechanism and enzymology of pyruvate dehydrogenation in Escherichia coli [6], only a little information is available about the pyruvate dissimilation. The elucidation of its mechanism has been hampered mainly by the oxygen inhibition of the system [7,8] and the multiplicity of low and high molecular weight

Pyruvate Formate‐Lyase of Escherichia coli: the Acetyl‐Enzyme Intermediate

“…Thiamine coenzyme could not be detected in the enzyme preparations [3]. Making use of the now known molar stoichiometry, these analyses have recently been repeated with a confirmation of the earlier results.…”

“…It is therefore suggested that this compound (and the natural pyruvate) exerts a regulatory role as a complexing ligand, most probably of the catalytical enzyme I1 fraction. Such a role has also been suggested for adenosylmethionine [3]. It was excluded that this molecule or any part of it is a constituent of the active pyruvate formate-lyase species (unpublished experiments, 1972, performed in conjunction with K. Jungermann and R. Thauer, University of Freiburg).…”

“…With this reduction system and employing an optical assay described below, which enabled rapid monitoring of the active enzyme species appearing, it was to be recognized that there is a virtual absolute requirement of pyruvate in the activation process in addition to the earlier found total requirement of S-adenosylmethionine [3,8]. It was subsequently detected that pyruvate was replaceable by its analogue oxamate ( Table 1).…”

“…The inactive form of pyruvate formate-lyase (formerly designated as 'enzyme I'), was purified from cell extracts of aerobically grown Escherichia coli K 12 through the hydroxyapatite-step, and 'enzyme 11 ' (catalyzing the pyruvate formate-lyase activation) through the DEAE-cellulose step by procedures slightly modified from that described previously [3] (see also [5]). Flavodoxin was isolated as described previously [4].…”

“…Pyruvate formate-lyase activity formed was measured by the optical assay ( Fig. 2A) from aliquots withdrawn with an automatic pipet of maximal activity of purified fractions of the Fe2+-dependent enzyme 11; see [3].) Next, the contents of the two arms were mixed and the tube was illuminated from a daylight lamp.…”