Coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme B₁₂-dependent mutase

Abstract

Hydrogen transfer reactions catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase have very large kinetic isotope effects, indicating that they proceed by a highly quantal tunneling mechanism. We explain the kinetic isotope effect by using a combined quantum mechanical/molecular mechanical potential and semiclassical quantum dynamics calculations. Multidimensional tunneling increases the magnitude of the calculated intrinsic hydrogen kinetic isotope effect by a factor of 3.6 from 14 to 51, in excellent agreement with experimental results. These calculations confirm that tunneling contributions can be large enough to explain even a kinetic isotope effect >50, not because the barrier is unusually thin but because corner-cutting tunneling decreases the distance over which the system tunnels without a comparable increase in either the effective potential barrier or the effective mass for tunneling.enzyme kinetics ͉ kinetic isotope effects ͉ quantum dynamics ͉ molecular modeling ͉ ensemble-averaged variational transition state theory T he determination of the crystal structure of coenzyme B 12 or 5Ј-deoxyadenosylcobalamin (AdoCbl) by Lenhart and Hodgkin (1) revealed, unexpectedly at the time, a cobalt-carbon bond to C5Ј of the deoxyadenosyl ligand. Homolytic rupture of this bond is a key step in the carbon-skeletal rearrangements catalyzed by AdoCbl-dependent enzymes (2, 3), although atomistic details of the mechanism have remained elusive. B 12 -dependent rearrangements play vital roles in amino acid catabolism and fermentation, and methylmalonyl-CoA mutase (MMCM) is the only B 12 -dependent isomerase that is found in both mammals and bacteria. MMCM catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA, which then can enter the Krebs cycle. An anomalously large kinetic isotope effect (KIE) has been observed for the hydrogen atom transfer from substrate to the deoxyadenosyl radical (dAdo⅐) in the step that initiates the isomerization reaction in the enzyme (4). This KIE poses a challenge to theoretical understanding (5), and we use it here as a key to elucidating atomistic details of the reaction catalyzed by MMCM.Recent advances in computational enzyme kinetics have allowed for a detailed understanding of how many enzymes work, and it now is appreciated that enzyme-catalyzed proton and hydride transfer reactions often occur by quantum mechanical tunneling (6, 7). Hydrogen atom transfers are more elusive because they often proceed by proton-coupled electron transfer. However, B 12 -dependent isomerases and reductases are believed to operate by radical translocation (3, 8-11), and various substrate and product radical intermediates have been detected by EPR spectroscopy in these enzymes. The observation of very large hydrogen/deuterium (H/D) KIEs for hydrogen radical transfer to or from dAdo⅐ in several enzymes (5, 12-15) raises the possibility that tunneling may be a common catalytic strategy for such translocation. Confirmation of this interpretation presently is best achieved by atomistic simulati...

Insights into the mechanisms of adenosylcobalamin (coenzyme B12)-dependent enzymes from rapid chemical quench experiments

“…This contrasts with much larger isotope effects (in the range of 30-50) measured for other B 12 enzymes such as methylmalonyl-CoA mutase [36][37][38][39] and B 12 model systems (measured by different techniques) that have been attributed to extensive hydrogen tunnelling. This argues strongly for a role for glutamate mutase in modulating the transition state for hydrogen transfer, and thereby changing the isotope effect.…”

The entropic contributions in vitamin B 12 enzymes still reflect the electrostatic paradigm

“…Furthermore, although ref. 40 seems to support the stepwise mechanism and was interpreted as such by some workers (e.g., ref. 18), no 2D concerted surface has been reported or examined.…”

“…Note also that ref. 40 used a semiempirical model that has not been calibrated by ab initio surfaces. Furthermore, although ref.…”

Theoretical studies on bimolecular reaction dynamics

“…These methods have been tested and checked for accuracy on the benchmark atom-diatom reactions (45) and are now being applied to reactions of biochemical importance. A good example is application to the coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme (46). Recently, a hybrid quantum-classical path integral method has also been applied effectively to this problem (47).…”