Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.
o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.
Bhattacharyya
D K
DK
Department of Biological Sciences, Northern Illinois University, DeKalb 60115, USA.
Kwon
O
O
Meganathan
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010654:Phenylbutyrates / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013389:Succinate-CoA Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D014812:Vitamin K / Q000096:biosynthesis*
D006898:Hydroxylamines; D010654:Phenylbutyrates; D014812:Vitamin K; C013691:o-succinylbenzoic acid; D019811:Hydroxylamine; D006639:Histidine; D000255:Adenosine Triphosphate; D013389:Succinate-CoA Ligases; C022693:O-succinylbenzoate - CoA ligase; D008274:Magnesium; D004047:Diethyl Pyrocarbonate