Enzymes of the beta-ketoadipate pathway in Pseudomonas putida: kinetic and magnetic resonance studies of the cis,cis-muconate cycloisomerase catalyzed reaction.

Steady-state kinetic analysis of the divalent metal ion requiring cis,cis-muconate cycloisomerase catalyzed interconversion of cis,cis-muconate and (+)-muconolactone obeys Michaelis-Menten kinetics and the Haldane relationship from pH 6.2 to 8.3. The pH vs. kcat/Km profiles suggest free-enzyme apparent pKa values of 6.2 and 7.4: the reciprocal behavior of the data with respect to the latter pKa value is consistent with base-acid catalysis by the enzyme involving proton removal from the lactone and protonation of cis,cis-muconate, respectively. This catalysis by the enzyme of proton transfer is consistent with the stereospecific incorporation of solvent deuterium into the pro-5R position of (+)-muconolactone in the enzyme-catalyzed reaction: in reverse, the departure of the carboxylic oxygen atom and proton from the C(4) and C(5) carbon atoms follows a syn (cis) route [Avigad, G., & Englard, S. (1969) Fed. Proc., Fed. Am. Soc. Exp. Biol. 28, 345, Abstr. 486]. The titration of enzyme freed of divalent metal ion with manganous ion, monitored by electron paramagnetic resonance spectroscopy and steady-state kinetic measurements, indicates a single binding site per subunit characterized by KdissE X Mn = [E] [Mn2+]/[E X Mn2+] = 4.5 and 3.0 microM, respectively, the latter value analyzed via a rapid equilibrium mechanism. The paramagnetic effects of Mn2+ on the 1/T1 and 1/T2 values for the H-5S proton of (+)-muconolactone in the E X ML X Mn ternary complex provide an estimate of the correlation time, tau c, at 5 X 10(-9) s from the T1/T2 ratio, indicating that the condition of rapid exchange of (+)-muconolactone in solution with the ternary complex obtains.(ABSTRACT TRUNCATED AT 250 WORDS)