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  • Title: Protonation of arginine 57 of Escherichia coli ornithine transcarbamoylase regulates substrate binding and turnover.
    Author: Goldsmith JO, Kuo LC.
    Journal: J Biol Chem; 1993 Sep 05; 268(25):18485-90. PubMed ID: 8395503.
    Abstract:
    The amino acid residue Arg57 of Escherichia coli ornithine transcarbamoylase is located in the carbamoyl phosphate-binding domain of the enzyme. This residue has been implicated to be critical for efficient carbamoylation and is linked to an induced-fit protein isomerization elicited by the lead substrate carbamoyl phosphate. To elucidate its role in substrate binding and catalysis, Arg57 has been substituted by one of two amino acids, glycine and histidine, varying in size and charge. Elimination of the positive charge and steric bulk on residue 57 with an arginine-to-glycine substitution results in an enzyme which binds its substrates in a random order (Kuo, L. C., Miller, A. W., Lee, S., and Kozuma, C. (1988) Biochemistry 27, 8823-8832). Replacement of Arg57 by histidine provides a substantial portion of the steric bulk at this residue position and also brings the pKa of this residue into an experimentally observable window. Kinetic and pH titration experiments reveal that when His57 is deprotonated the enzyme binds its substrates randomly. However, when His57 is protonated the enzyme observes the obligatory substrate binding order as seen for the wild type. Both the Gly57 and His57 point mutants are incapable of undergoing the carbamoyl phosphate-induced protein conformational changes apparent in the wild type. These results reveal that the induced-fit isomerization of ornithine transcarbamoylase does not contribute to the order in which the substrates bind. A comparison of the reaction schemes and pH profiles of the wildtype and His57 enzymes further indicates that protonation of residue 57 facilitates formation of the binary enzyme-carbamoyl phosphate complex and augments the turnover rate of the reaction. Together with steady-state kinetic parameters derived in terms of microscopic rate constants, our results provide additional support to our earlier suggestion (Zambidis, I. and Kuo, L. C. (1990) J. Biol. Chem. 265, 2620-2623) that the turnover rate of the wild-type ornithine transcarbamoylase in the forward reaction is largely dictated by the rate of the carbamoyl phosphate-induced isomerization.
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