Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage

Recently, Knappe and co-workers [Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., & Ganzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332] have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using [2H2]hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from [2H]formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.     

Possible involvement of acyltransferase systems in the formation of pulmonary surfactant lipid in rat

Dithiobis (2-nitrobenzoic acid)-resistant and -sensitive glycerophosphate acyltransferase systems were present in rat lung as in liver. The former was specific for palmitate while the latter could incorporate saturated and unsaturated acyl-CoAs comparably. The former has higher affinity for palmitate than the latter indicating that the 1-position of glycerophosphate can be acylated selectively with palmitate under certain conditions. The specificities of 1-acylglycerophosphate and 1-acylglycerophosphocholine acyltransferase systems were similar in lung and liver; both systems showed higher specificities for unsaturated acyl-CoAs. However, the selectivities observed at lower concentrations of phospholipid acceptors in the presence of equimolar mixtures of saturated and unsaturated acyl-CoAs were much different; the lung systems showed relatively higher selectivities for palmitate than the liver systems in the formation of both diacylglycerophosphate and phosphatidylcholine. On the other hand, palmitate was excluded almost completely from the 2-position in the 1-acylglycerophosphoethanolamine acyltransferase systems in lung and liver. These observations provide an enzymatic basis for describing the formation of pulmonary surfactant lipids in rat via acyltransferase systems.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.     

Identification of a cell cycle-dependent gene product as a sialic acid-binding protein

A Ca2+-dependent sialic acid-binding protein was purified on fetuin-Sepharose from various types of human tissue. The molecular mass was determined to be 10,315 Da by laser desorption mass spectrometry. Partial sequence analysis after cyanogen bromide cleavage that yielded one N-terminus accessible for Edman degradation revealed an identity to an internal stretch following the only methionine residue within a putative amino acid sequence (Mr 10,048), deduced from the cDNA of a cell cycle-specific gene. The reported biochemical identification is a prerequisite to infer the biological role of the so far undetected gene product. Initial glycohistochemical studies with sialic acid-(BSA-biotin) raised evidence for nuclear localization of sialic acid-binding sites that might reflect, at least in part, detection of this protein.