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Nitrate reductase in E. coli: Properties of the enzyme and in vitro reconstitution from enzyme-deficient mutants

1974; Wiley; Volume: 2; Issue: 5-6 Linguagem: Inglês

10.1002/jss.400020515

1547-9366

C H MacGregor, Carl A. Schnaitman,

Enzyme Structure and Function

Abstract The nitrate reductase of E. coli is an inducible membrane protein with a molecular weight of about 800,000. The enzyme consists of four subunits of 60,000 molecular weight, four subunits of 142,000 molecular weight, four molecules of molybdenum, and nonheme iron. The enzyme may be solubilized by heat extraction, which results from limited digestion by a membrane‐bound protease, or by Triton X‐1 00. When the enzyme is isolated from Triton‐solubilized cytoplasmic membrane by immune precipitation, it contains a third protein of 20,000 molecular weight which may be a cytochrome. Chlorate resistant (chl) mutants of E. coli lack functional nitrate reductase. Mutants of the classes (chl)and chlB have all of the enzyme polypeptides present in the membrane JI intact form, while in classes chlC and chlE the membrane contains degraded fragments of the polypeptides, suggesting proteolysis of a defective enzyme. Reconstitution of nitrate reductase activity occurs when soluble extracts of various classes of mutants are mixed and incubated at 32°C. This reconstitution requires three things: (a) intact enzyme polypeptides in the form of small soluble lipoprotein fragments resulting from fragmentation of the cytoplasmic membrane during cell breakage; (b) a molybdenum factor which is present in the wild‐type membrane and which accumulates in the cytoplasm of chlB mutants in soluble form; and (c) a soluble factor or enzyme, presumably the chlB gene product, which adds the molybdenum factor to the enzyme Two conclusions may be drawn from these observations. First, the enzyme is bound t o the membrane by small, hydrophobic regions on one or more of the subunits. Second, the process of reconstitution from mutant extracts is different from the process involved in de novo synthesis of the enzyme in wild‐type E. coli.