Stabilization of Radical Intermediates by an Active-Site Tyrosine Residue in Methylmalonyl-CoA Mutase^,

Abstract

The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall release of tritium was four times faster when succinyl-CoA was used as substrate. The tritium isotope effect on the enzyme catalyzed hydrogen transfer, measured with methylmalonyl-CoA as a substrate, was kH/kT = 30, which is within the expected range for a full primary kinetic tritium isotope effect. The different partitioning of tritium, dependent upon which substrate was used, and the normal value for the kinetic tritium isotope effect contrast markedly with the behavior of wild-type mutase. It appears that the loss of a single interaction involving the hydroxyl group of tyrosine 89 both affects the stability of radical intermediates and decreases the rate of interconversion of the substrate- and product-derived radicals.

Changes in the free energy profile of glutamate mutase imparted by the mutation of an active site arginine residue to lysine

“…For wild-type MMCM, tritium partitioning is independent of whether methylmalonyl-CoA or succinyl-CoA is the substrate, indicating that rearrangement of the substrate radicals is rapid compared with hydrogen transfer. However, two active site mutations, His244Gln and Tyr248Phe have been identified which cause tritium in AdoCbl to preferentially partition back to the substrate [29,30]. These mutations appear to exert effects very similarl to what we observe in the glutamate mutase Arg100Lys mutant.…”

“…In particular, we recently described the effects of mutating Arg-100, which forms a salt bridge with the γ-carboxylate of the substrate (Fig. 2) [28][29][30]. We compare the results of our present studies with the previous data, which affords some insights into the role of active site residues in promoting the rearrangement of substrate radicals in these enzymes.…”

“…The partitioning of hydrogen isotopes from an enzymebound intermediate between products and substrates provides an elegant way of investigating the relative heights of energetic barriers in an enzyme-catalyzed reaction. We have previously applied the same technique to investigate the partitioning of tritium at the 5'-carbon of AdoCbl between substrate and product in the wild-type enzyme [27]; tritium partition experiments have also been used to investigate the free energy profile of AdoCbl-dependent methylmalonyl-CoA mutase [28][29][30]. We compare the results of our present studies with the previous data, which affords some insights into the role of active site residues in promoting the rearrangement of substrate radicals in these enzymes.…”

Alternative Pathways for Radical Dissipation in an Active Site Mutant of B12-Dependent Methylmalonyl-CoA Mutase

“…computational studies on methylmalonyl-CoA mutase have begun to provide insights into these issues (3)(4)(5)(6)(7)(8). They point to the critical role played by individual active site residues in labilizing the Co-carbon bond (7), and in shielding radical intermediates from unwanted side-reactions (4,6,8).…”

“…The hypothesis that Y89 functions as a molecular wedge that labilizes the Co-carbon bond, was derived from crystallographic data, which revealed that substrate-driven barrel closure results in a large motion of this residue and steric crowding above the corrin ring (9,11). The role of Y89 in labilizing the Co-carbon bond was supported by mutagenesis and kinetic studies, which also revealed its contribution to the rearrangement step (3,7).…”