Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis.
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Kuo LC, Miller AW, Lee S, Kozuma C
Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis.
Biochemistry. 1988 Nov 29;27(24):8823-32.
- PubMed ID
- 3072022 [ View in PubMed]
- Abstract
In the carbamoyl-transfer reaction catalyzed by ornithine transcarbamoylase, an arginine residue in the active site of the Escherichia coli enzyme has been suggested to bind the phosphate moiety of the substrate carbamoyl phosphate. With the application of site-specific mutagenesis, the most likely arginine residue among three candidates at the binding site of carbamoyl phosphate, Arg-57, has been replaced with a glycine. The resultant Gly-57 mutant enzyme is drastically inefficient in catalysis. In the synthesis of L-citrulline from carbamoyl phosphate and L-ornithine with the release of inorganic phosphate, the turnover rate of the mutant is 21,000-fold lower than that of the wild type. However, the mutation of Arg-57 affects only moderately the binding of carbamoyl phosphate; the dissociation constant of this substrate, measured under steady-state turnover condition, is increased from 0.046 to 3.2 mM by the mutation. On the other hand, ornithine binding is substantially affected as estimated by the change in the dissociation constant of its analogue L-norvaline. The dissociation constant of L-norvaline increases about 500-fold from 54 microM for the wild type to 25 mM for the mutant. Since Arg-57 is expected to be distal from the ornithine site and the amino acid (both ornithine and norvaline) binds only after carbamoyl phosphate in the wild-type reaction, the poor norvaline affinity to the mutant suggests that Arg-57 is involved in interactions essential for productive addition of the amino acid. This interpretation is supported by difference ultraviolet absorption spectra which show that the conformational changes induced in the wild type by carbamoyl phosphate upon binding are absent in the mutant. Furthermore, steady-state kinetic data reveal that the ordered binding mechanism of the wild-type enzyme is transformed into a random binding mechanism in the mutant. Thus, the presence of carbamoyl phosphate in the mutant active site is no longer a requisite for ornithine binding. In the 5-50 degrees C temperature range, transcarbamoylation catalyzed by either the wild type or the mutant observes the Arrhenius rate law with almost identical enthalpies of activation, 11 and 10 kcal/mol, respectively. The entropy of activation is -5.5 eu for the wild-type reaction and -29 eu for the mutant reaction, accounting for a loss of 6-7 kcal/mol in the rate-determining step of the enzymic reaction.(ABSTRACT TRUNCATED AT 400 WORDS)