Purification and Properties of the Wild Type and a Feedback-resistant Phosphoribosyladenosine Triphosphate: PYROPHOSPHATE PHOSPHORIBOSYLTRANSFERASE, THE FIRST ENZYME OF HISTIDINE BIOSYNTHESIS IN SALMONELLA TYPHIMURIUM

Abstract

Abstract A purification procedure has been devised for phosphoribosyltransferase, the l-histidine-sensitive first enzyme in the pathway for histidine biosynthesis in Salmonella typhimurium. The procedure was applied to a wild type and a feedback-resistant strain. The enzymes from both strains appeared nearly homogeneous in the ultracentrifuge and upon polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate. The enzymes had similar sedimentation coefficients in the ultracentrifuge and similar mobilities on polyacrylamide gels containing sodium dodecyl sulfate. Tryptic peptide maps of the two enzymes could not be distinguished. The wild type enzyme gave regular Michaelis-Menten kinetics but initial velocity analysis at a constant optimal magnesium to ATP ratio (2:1) gave nonparallel lines on double reciprocal plots. l-Histidine was an uncompetitive inhibitor with respect to phosphoribosyl pyrophosphate, while it was a noncompetitive inhibitor with respect to ATP. The curves for l-histidine and l-thiazolealanine inhibition were sigmoid in shape, and conversion to Hill plots gave straight lines with slopes of 1.6 and 1.8, respectively. Inhibition by both effectors was pH-dependent. The reverse reaction was also inhibited by l-histidine. A difference spectrum of the wild type enzyme showed a striking increase in absorbance at 280 mµ upon the addition of l-histidine, whereas that of the feedback-resistant enzyme remained constant following the addition of l-histidine.