Role of Active Site Residues in Promoting Cobalt–Carbon Bond Homolysis in Adenosylcobalamin-Dependent Mutases Revealed through Experiment and Computation

Abstract

Adenosylcobalamin serves as a source of reactive free radicals that are generated by homolytic scission of the coenzyme’s cobalt-carbon bond. AdoCbl-dependent enzymes accelerate AdoCbl homolysis by ~1012-fold, but the mechanism by which this is accomplished remains unclear. We have combined experimental and computational approaches to gain molecular-level insight into this process for glutamate mutase. Two residues, glutamate-330 and lysine-326, form hydrogen bonds with the adenosyl group of the coenzyme. A series of mutations were introduced at these positions that impair the enzyme’s ability to catalyze coenzyme homolysis and tritium exchange with the substrate by 2 – 4 orders of magnitude. These mutations, together with the wild-type enzyme, were also characterized in silico by molecular dynamics simulations of the enzyme:AdoCbl:substrate with AdoCbl modeled in either the associated (Co-C bond formed) or the dissociated (adenosyl radical + CblII) state. The simulations reveal that the number of hydrogen bonds between the adenosyl group and the protein side-chains increases in the homolytically-dissociated state, with respect to the associated state, for both the wild-type and mutant enzymes. The mutations also cause a progressive increase in the mean distance between the 5′-carbon of the adenosyl radical and the abstractable hydrogen of the substrate. Interestingly, the distance between the 5′-carbon and substrate hydrogen, determined computationally, was found to inversely correlate with the logk for tritium exchange (r = 0.93) determined experimentally. Taken together, these results point to a dual role for these residues: they both stabilize the homolytic state through electrostatic interactions between the protein and the dissociated coenzyme, and correctly position the adenosyl radical to facilitate hydrogen abstraction from the substrate.

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

“…Other AdoCbl-dependent mutases similarly position their substrates at the same distance from the Cbl, as determined from crystal structures (24,29,33) and by electron paramagnetic resonance spectroscopic studies of glutamate mutase (62) and MCM (63) under catalytic conditions. Pseudorotation of the 5Ј-dAdo ribose has also been suggested from structural and biochemical studies of glutamate mutase (33,55) and from computational studies on MCM (64). Thus, given the combined structural, biochemical, and computational evidence, it appears that this mechanism of moving the active C5Ј radical toward substrate is conserved in AdoCbl-dependent mutases such as acyl-CoA mutases.…”

“…To accelerate Co-C bond homolysis, substrate binding likely modulates the interactions between the protein and the 5Ј-dAdo, for example by inducing large scale conformational changes such as the TIM barrel motions or by altering active site electrostatics or dynamics to destabilize the C2Ј-endo form or to stabilize the C3Ј-endo form (55,68,69). It is unclear how many molecular mechanisms AdoCbl enzymes use to afford the substantial 10 12 enhancement in Co-C bond homolysis that accompanies substrate binding (1,3,70).…”

“…6, c and d) (33). Glu-905 is also conserved in glutamate mutase as Glu-330, and mutation of this Glu-330 to Asp, Gln, or Ala leads to a drastic reduction in activity, supporting its importance for catalysis (55).…”

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

“…It was also shown that the assessment of the steric effect could not be done without using converging freeenergy calculations rather than energy minimization approaches. The important finding that the interactions between the 2′-and 3′-OH groups of the ribose with Glu-370 play a major role is also supported by the reduced activity upon removal of the 2′-OH group (24) and the loss of activity upon mutation of the equivalent Glu-330 in glutamate mutase (25). The above discussion is basically a summary of what we have already established, whereas the focus of the current work is on the origin of the observed entropic effect.…”

Synthesis and Characterization of 4‐, 5‐, and 6‐Coordinate Tris(1‐ethyl‐4‐isopropylimidazolyl‐κN)phosphine Cobalt(II) Complexes

“…[1][2][3][4] Cobalt can also be substituted for zinc in zinc(II) enzymes to make the still-functioning enzymes spectroscopically active (UV/Vis, magnetic circular dichroism, NMR, and electron paramagnetic resonance spectroscopy). [5][6][7][8] The large number of zinc enzymes provides ample opportunity for these types of substitutions.…”