A new and highly sensitive assay for the ATP phosphoribosyltransferase that catalyzes the first step of histidine biosynthesis

✍ Scribed by Henry M. Kronenberg; Tikvah Vogel; Robert F. Goldberger

• Publisher: Elsevier Science
• Year: 1975
• Tongue: English
• Weight: 500 KB
• Volume: 65
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(75)90522-9

✦ Synopsis

A new method is described for assaying the ATP phosphoribosyltransferase (EC 1.4.2.17) that catalyzes the first step of histidine biosynthesis in bacterial cells. This is a highly sensitive radioassay, capable of detecting fewer than lOy molecules of enzyme. In contrast to the previously available assay, it can be carried out in the presence of other molecules that absorb uv light. The assay may find useful application in studies on the in vitro synthesis of the enzymes for histidine biosynthesis and in studies on the various aggregation states of the enzyme.

The first enzyme in the pathway for histidine biosynthesis in Salmonella typhimuriwn, N-l-(5'-phosphoribosyl)adenosine triphosphate: pyrophosphate phosphoribosyltransferase (EC 2.4.2.17, ATP phosphoribosyltransferase), catalyzes the reaction:

This enzyme controls histidine biosynthesis in two ways: (1) the activity of the enzyme is inhibited by L-histidine, providing a feedback mechanism that allows the end product to regulate the flow of substrate into the biosynthetic pathway (1); and (2) the enzyme itself helps regulate expression of the histidine operon in a manner that is not yet clear (for review, see ref.

2). The enzyme is a hexamer, with a molecular weight of 215,000 (3). It has sites specific for binding the two substrates. ATP and PRPP, L-histidine (4), histidyl-tRNA (5), and the DNA of the histidine operon (M. Meyers, T. Vogel, and R. Goldberger, in preparation). The only previously described assay for the enzyme monitors increase in absorbancy at 290 nm caused by attachment of a phosphoribosyl moiety to the N-1 of adenine (1).