Are the hydrogens removed simultaneously or one by

Are the hydrogens removed simultaneously or one by one? — Δ1-KSTDs can catalyze the exchange of alkali-labile tritium or deuterium atoms at the C2 Flutamide of their substrates, even when enzyme turnover was prevented by the absence of an electron acceptor for the oxidative half-reaction [97,98] or by keeping the flavin prosthetic group in the reduced state [96]. This observation indicates that the enzymes more likely employ a stepwise unimolecular elimination conjugate base (E1cB) mechanism, in which departure of the first hydrogen atom precedes that of the second hydrogen atom. Such a mechanism requires the formation of an intermediate. A concerted bimolecular elimination (E2) mechanism, in which the two hydrogens depart simultaneously without the formation of an intermediate, is less likely. Thus, 1(2)-dehydrogenation by Δ1-KSTD has been considered to involve a two-step mechanism, i.e. an initial fast step followed by a slow rate-determining step [98]. The fast step was proposed to be initiated by an interaction of the C3 carbonyl group of the 3-ketosteroid substrate with an electrophile. This interaction stimulates labilization of the C2 hydrogen atoms. Subsequent abstraction of a proton from this atom by a general base results in either an enolate [97,98] or a carbanionic [96] intermediate. In the slow step, a double bond is proposed to be formed between the C1 and C2 atoms when a hydride ion is transferred from the C1 atom of the intermediate to the flavin prosthetic group [96,97,98]. This proposed step-wise mechanism is in contrast to the concerted removal of the hydrogens catalyzed by acyl coenzyme A dehydrogenases [133,134]. — The nature and positions of the amino-acid residues involved in catalysis by Δ1-KSTD were clarified with the structure determination of the Δ1-KSTD1•ADD complex combined with mutational studies on the enzyme. A superposition of the structure of the substrate AD (8) on that of the product ADD (9) as bound in the Δ1-KSTD1•ADD complex structure revealed that 1) the hydroxyl group of Tyr-487 and the backbone amide of Gly-491 would be at the right positions for hydrogen bond formation with the C3 carbonyl group of the substrate; 2) the hydroxyl group of Tyr-318 would be at ˜3.0 Å from the C2 atom of the substrate; 3) the N5 atom of the FAD isoalloxazine ring would be at ˜2.6 Å from the C1 atom of the substrate; and 4) the isoalloxazine ring and Tyr-318 would be on opposite sides of the A-ring of the substrate, with the isoalloxazine ring at the α-side and Tyr-318 at the β-side. Thus, the substrate would be bound in the active site such that its C1 and C2 atoms are positioned appropriately for hydride and proton abstraction, respectively [30]. These observations facilitated a detailed description of the 1(2)-dehydrogenation mechanism of Δ1-KSTD1 (Fig. 4) [30]. Tyr-487 and Gly-491 tightly bind the carbonyl oxygen of the 3-ketosteroid substrate to promote keto-enol tautomerization and labilization of the C2 hydrogen atoms. The hydroxyl group of Tyr-318 serves as a general base that abstracts the axial β-hydrogen from the C2 atom as a proton. A transient carbanionic intermediate, which is most likely stabilized by keto-enol tautomerization, is formed. This negatively charged intermediate can be stabilized by the delocalization of its charge over the C3 keto group and the interaction with the positive N-terminal helix macro-dipole of a nearby α-helix. Tyr-119, whose hydroxyl group is hydrogen bonded to the Tyr-318 hydroxyl group, may increase the basic character of Tyr-318 and facilitate proton relay to the solvent. The negative charge of the intermediate is then shifted to the C1 atom to form the double bond between the C1 and C2 atoms. In synchrony, the N5 atom of the FAD prosthetic group abstracts the axial α-hydrogen from the C1 atom as a hydride ion, generating a reduced anionic FAD. The negative charge of this anion can be delocalized over the pyrimidine moiety of the isoalloxazine prosthetic group. The pyrimidine moiety is stabilized by hydrogen bonding interactions with the protein backbone as well as by the helix macro-dipole interaction with the N-terminal end of an α-helix. Re-oxidation of the reduced FAD by an electron acceptor in the subsequent oxidative half-reaction will complete the catalytic cycle and make the enzyme available for another cycle [30].