Divergent mechanisms of suicide inactivation for ethanolamine ammonia-lyase

Ab initio molecular orbital calculations have been used to study the mechanism of suicide inactivation of ethanolamine ammonia-lyase induced by three different substrate analogues. Analysis of the normal catalytic mechanism with 2-aminoethanol (ethanolamine) as substrate predicts that both the hydrogen-abstraction and hydrogen-reabstraction steps involving the B 12-cofactor are likely to be exothermic. On the other hand, the proposed inactivation mechanism for the first substrate analogue, glycolaldehyde, leads to a highly stabilized radical that results in a very endothermic (by ca. 90 kJ mol-1) hydrogen-reabstraction step, which is thought to halt the normal function of the enzyme. Curiously, the energy requirements for a catalytically imposed mechanism in the case of the second substrate analogue, 2-hydroxyethylhydrazine (HEH), parallel those for the catalytic substrate, despite the fact that HEH is found to inactivate EAL experimentally. However, further analysis reveals the presence of a lower energy pathway for HEH that leads to the formation of the highly stabilized hydrazinium radical cation. In a manner similar to when glycolaldehyde is the substrate analogue, this results in an endothermicity for the hydrogen-reabstraction step that is prohibitively large. In contrast to these related inactivation mechanisms, the third substrate analogue, 2-aminoacetaldehyde, apparently accomplishes the inactivation of EAL in an entirely different manner. A pathway for the experimentally observed formation of acetic acid and ammonium cation has been identified and appears catalytic in the sense that 5′-deoxyadenosyl radical is regenerated. However, mechanisms to account for the subsequent formation of 4′,5′- anhydroadenosine and degradation of the corrinoid ring of the cofactor have not been elucidated

In search of radical intermediates in the reactions catalyzed by lysine 2,3-aminomutase and lysine 5,6-aminomutase

High-level ab initio calculations have been used to study radical intermediates in the reactions catalyzed by lysine 2,3-aminomutase (2,3-LAM) and lysine 5,6-aminomutase (5,6-LAM). The reactions of these enzymes with the substrate analogues 4-oxalysine (4-OL), 4-thialysine (4-TL), or trans-4,5-dehydrolysine (t-4,5-DL) are rationalized in terms of stabilization provided by the substituent to the adjacent radical center. Large changes in the exothermicity accompanying the initial H-abstraction are observed relative to the lysine reference values that follow the series 4-OL < 4-TL < t-4,5-DL. These changes have the primary effect of increasing the endothermicity for subsequent ring-closure to form the putative aziridinylcarbinyl radical intermediate. Such stabilization is consistent with experimental observations of the substrate-derived radical (S•) in the reaction of 2,3-LAM with 4-TL as well as the ability of t-4,5-DL to act as an irreversible inhibitor of 2,3-LAM. Our calculations suggest that 4-TL and trans-3,4-dehydrolysine may also permit experimental characterization of S• radicals in the reactions catalyzed by 5,6-LAM. Strategies for modifying PLP are presented that might lead to the first observation of the aziridinylcarbinyl radical intermediate (I•) in the aminomutase-catalyzed reactions

Suicide Inactivation of Dioldehydratase by Glycolaldehyde and Chloroacetaldehyde: an Examinaton of the Reaction Mechanism

High-level ab initio calculations have been used to study the mechanism for the inactivation of diol dehydratase (DDH) by glycolaldehyde or 2-chloroacetaldehyde. As in the case of the catalytic substrates of DDH, e.g., ethane-1,2-diol, the 5′-deoxyaden

Toward an improved understanding of the glutamate mutase system

High-level quantum chemistry calculations have been used to examine the catalytic reactions of adenosylcobalamin-dependent glutamate mutase (GM) with the natural substrate (S)-glutamic acid. We have also examined the rearrangement of (S)-2-hydroxyglutaric acid, (S)-2-thiolglutaric acid, and 2-ketoglutaric acid, all of which have previously been shown to react as substrates or inhibitors of the enzyme. Our calculations support the notion that the 100-fold difference in kcat between glutamate and 2-hydroxyglutarate is associated with the relatively high energy of the glycolyl radical intermediate compared with the glycyl radical. More generally, calculations of radical stabilization energies for a variety of substituted glycyl radical analogues indicate that modifications at the radical center can profoundly affect the relative stability of the resulting radical, leading to important mechanistic consequences. We find that the formation of a thioglycolyl radical, derived from (S)-2-thiolglutaric acid, is highly dependent on the protonation state of sulfur. The neutral radical is found to be of stability similar to that of the glycolyl radical, whereas the S- form of the thioglycolyl radical is much more stable, thus providing a rationalization for the inhibition of the enzyme by the substrate analogue 2-thiolglutarate. Two possible rearrangement pathways have been examined for the reaction of GM with 2-ketoglutaric acid, for which previous experiments had suggested no rearrangement took place. The fragmentation-recombination pathway is associated with a fragmentation step that is very endothermic (by 102.2 kJ mol-1). In contrast, the addition-elimination pathway has significantly lower energy requirements. An alternative possibility, namely, that 2-ketoglutaric acid is bound in its hydrated form, 2,2-dihydroxyglutaric acid, also leads to a pathway with relatively low energy requirements, suggesting that some rearrangement might be expected under such circumstances

Insights into the Hydrogen-Abstraction Reactions of Diol Dehydratase: Relevance to the Catalytic Mechanism and Suicide Inactivation

High-level quantum chemistry calculations have been used to examine the hydrogen-abstraction reactions of diol dehydratase (DDH) in the context of both the catalytic mechanism and the enzyme dysfunction phenomenon termed suicide inactivation. The barriers for the catalytic hydrogen-abstraction reactions of ethane-1,2-diol and propane-1,2-diol are examined in isolation, as well as in the presence of various Bransted acids and bases. Modest changes in the magnitudes of the initial and final abstraction barriers are seen, depending on the strength of the acid or base, and on whether these effects are considered individually or together. The most significant changes (ca. 20 kJ mol -1) are found for the initial abstraction barrier when the spectator OH group is partially deprotonated. Kinetic isotope effects including Eckart tunneling corrections (KIEs) have also been calculated for these model systems. We find that contributions from tunneling are of a magnitude similar to that of the contributions from semiclassical theory alone, meaning that quantum effects serve to significantly accelerate the rate of hydrogen transfer. The calculated KIEs for the partially deprotonated system are in qualitative agreement with experimentally determined values. In complementary investigations, the ability of DDH to become deactivated by certain substrate analogues is examined. In all cases, the formation of a stable radical intermediate causes the hydrogen re-abstraction step to become an extremely endothermic process. The consequent inability of 5′-deoxyadenosyl radical to be regenerated breaks the catalytic cycle, resulting in the suicide inactivation of DDH

Broken-Symmetry DFT Computations for the Reaction Pathway of IspH, an Iron-Sulfur Enzyme in Pathogenic Bacteria.

The recently discovered methylerythritol phosphate (MEP) pathway provides new targets for the development of antibacterial and antimalarial drugs. In the final step of the MEP pathway, the [4Fe-4S] IspH protein catalyzes the 2e(-)/2H(+) reductive dehydroxylation of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) to afford the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Recent experiments have attempted to elucidate the IspH catalytic mechanism to drive inhibitor development. Two competing mechanisms have recently emerged, differentiated by their proposed HMBPP binding modes upon 1e(-) reduction of the [4Fe-4S] cluster: (1) a Birch reduction mechanism, in which HMBPP remains bound to the [4Fe-4S] cluster through its terminal C4-OH group (ROH-bound) until the -OH is cleaved as water; and (2) an organometallic mechanism, in which the C4-OH group rotates away from the [4Fe-4S] cluster, allowing the HMBPP olefin group to form a metallacycle complex with the apical iron (η(2)-bound). We perform broken-symmetry density functional theory computations to assess the energies and reduction potentials associated with the ROH- and η(2)-bound states implicated by these competing mechanisms. Reduction potentials obtained for ROH-bound states are more negative (-1.4 to -1.0 V) than what is typically expected of [4Fe-4S] ferredoxin proteins. Instead, we find that η(2)-bound states are lower in energy than ROH-bound states when the [4Fe-4S] cluster is 1e(-) reduced. Furthermore, η(2)-bound states can already be generated in the oxidized state, yielding reduction potentials of ca. -700 mV when electron addition occurs after rotation of the HMBPP C4-OH group. We demonstrate that such η(2)-bound states are kinetically accessible both when the IspH [4Fe-4S] cluster is oxidized and 1e(-) reduced. The energetically preferred pathway gives 1e(-) reduction of the cluster after substrate conformational change, generating the 1e(-) reduced intermediate proposed in the organometallic mechanism

The Elusive 5′-Deoxyadenosyl Radical in Coenzyme-B₁₂-Mediated Reactions

Vitamin B₁₂ and its biologically active counterparts possess the only examples of carbon–cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B₁₂ (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car–Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5′-deoxyadenosyl radical (dAdo^•) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B₁₂ enzymes catalyze coenzyme activation and control highly reactive radical intermediates