Enzymes of the .beta.-ketoadipate pathway in Pseudomonas putida: primary and secondary kinetic and equilibrium deuterium isotope effects upon the interconversion of (+)-muconolactone to cis,cis-muconate catalyzed by cis,cis-muconate cycloisomerase

Authors:
  • KNKa Leung Ngai
    Ka Leung Ngai
  • RKRoland G. Kallen
    Roland G. Kallen
Publisher website
Google Scholar
Abstract
Primary and secondary kinetic and secondary equilibrium 2H isotope effect studies on the cis,cis-muconate cycloisomerase catalyzed interconversion of cis,cis-muconate(CCM) and (+)-muconolactone (ML) were performed. The primary and solvent kinetic 2H isotope effects on Vmax for the reactions of (+)-[5R-2H]muconolactone in water and ML in deuterium oxide to form CCM are about 2.5-2.6 with the heavier isotopic reactions being the slower ones. The secondary equilibrium isotope effect for the formation cis,-cis-[2,3,4,5-2H4]muconate from (+)-[2,3,4,5S-2H4]muconolactone is 1.32 for KH/KD = ([CCM-]/[ML])/([cis,cis-[2,3,4,5-2H4]muconate]/[(+)-[2,3,4,5S-2H4]muconolactone]) and agrees well with the measured value of 1.45 on the basis of the fumarase reaction. The secondary kinetic 3H isotope effect determined by the equilibrium perturbation method for the conversion of cis,cis-[2,3,4,5-2H4]muconate to (+)-[2,3,4,5S-2H4]muconolactone is 0.66, expressed as (VmaxCCM(H)/KmCCM(H))/(VmaxCCM(D)/KmCCM(D)). From the equilibrium and kinetic secondary 2H isotope effects, the calculated value for the kinetic secondary 2H isotope effect for the reverse reaction, (VmaxML(H)/KmML(H))/(VmaxML(D))/KmML(D), is .apprx. 0.96. The presence of a primary kinetic isotope effect with the absence of a secondary kinetic isotope effect with the absence of ML with CCM cycloisomerase to form CCM is most consistent with a stepwise mechanism involving the rate-determining generation of a carbanion intermediate from the ternary enzyme-Mn2+-ML complex. Concerted or stepwise mechanisms involving carbonium ion or covalent intermediates are therefore not likely for this enzyme-catalyzed syn (cis) elimination reaction.

This publication has 8 references indexed in Scilit: