Protonation of arginine 57 of Escherichia coli ornithine transcarbamoylase regulates substrate binding and turnover.

J.O Goldsmith,L.C Kuo

doi:10.1016/s0021-9258(17)46652-x

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.

Highlights

The amino acid residue A r g 7 of Escherichiacoli Ordered binding of substrates to enzymes has often been ornithine transcarbamoylase is located in the carba- suggested to result from conformational alterations of the moyl phosphate-binding domain of the enzyme
In the case of residue hasbeen implicated to be critical for efficient Escherichia coli ornithine transcarbamoylase, which catalyzes carbamoylation and is linked toan induced-fit protein the transfer of the carbamoyl group from carbamoyl phosisomerization elicited by the leadsubstrate carbamoyl phate to L-ornithine, the enzyme follows an ordered Bi Bi phosphate
The active-site histidine consistent with data obtained at pH 8.5 and 25 "C is shown of several monoisopropylphosphorylated serine proteases dein Scheme 1, where E, cp, and orn denote the free enzyme, protonate with a pK, between 9.7-11.4 while the same histicarbamoyl phosphate andL-ornithine, respectively

Summary

RESULTS

Michaelis constant (IC@'), and the pseudo second-order rate constant (&t/K~Lpob)t,ained at 25 "Cand at pH 8.5 and 11, X. Factor of -530, and a drop in the affinity for the bisubstrate His enzyme are shown in Fig. 1,and theresults are compiled analog, PALO, by a factor of -100. Results for the wild-type and Gly6' enzymes, between theArg[7] Gly and Arg7 + His enzymes with obtained from a previous report at pH 8.5 (6, 7) are respect to their affinities to the substrate, product, inhibitor, tabulated for comparison.The inhibition patterns of the Hiss[7] or bisubatrate analog. From uncompetitive to noncompetitive against carbamoyl phosphate for the His67enzyme (Fig.1B).There isno change in the inhibition patterns for the wild-type andthe Glg[7] norvaline; orn, L-ornithine; PALO, N-phosphonacetyl-L-ornithine; enzymes at pH 11from those at pH 8.5 Wild-typeand mutant inhibition patterns of ornithine transcarbamoylase with L-norvaline

Type of inhibition"

DISCUSSION

The extended or partially extended structure of the side chain