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An appropriately positioned general base helps remove the proton from the serine residue hydroxyl, increasing its nucleophilicity. A lysine residue, such as Lys73 in the class A beta-lactamase catalytic network, is a plausible participant; its protonation state depends on its active-site environment and interactions with other residues. A glutamate residue, notably Glu166, also participates in proton transfer and activation of the water needed for deacylation. Efficient catalysis requires proton-transfer chemistry as well as positioning the attacking oxygen. The precise division of roles during acylation depends on the enzyme state, so the drawing alone cannot assign a complete mechanism to one residue.^[ref]

At pH 7, a glutamate residue is normally carboxylate. Its most natural role is general-base activation of the attacking water. Draw the carboxylate oxygen lone pair toward a water H, the water O–H pair toward its oxygen, and that oxygen's lone pair toward C3 while the conjugated pi system moves toward the carbonyl oxygen:This can be concerted, avoiding a freely diffusing hydroxide intermediate. The protonated glutamate subsequently returns its proton to the enolate system, directly or through solvent, regenerating carboxylate. A favorable arrangement is needed; ordinary carboxylate does not quantitatively deprotonate bulk water in a prior equilibrium.

The side-chain amide of glutamine is neutral. Its N–H can donate a hydrogen bond to the carbonyl oxygen and stabilize developing enolate charge, while its amide oxygen can accept a hydrogen bond to orient attacking water. Draw $\mathrm{Gln}-\mathrm{CON}{\mathrm{H}}_2\cdots \mathrm{O}=C1-C2=C3$, with the dotted interaction from amide N–H to substrate oxygen. This assists polarization and transition state stabilization; the amide nitrogen is not an ordinary amine base because its lone pair is delocalized into its own carbonyl.

The guanidinium group of arginine is positively charged. It can stabilize developing negative charge by hydrogen bonding and electrostatic attraction, drawn $\mathrm{Arg}-\mathrm{guanidiniu}{\mathrm{m}}^{+}\cdots {\mathrm{O}}^{\delta -}-C1$. Its delocalized cation is not the preferred neutral general base at pH 7. Productive catalysis requires stronger stabilization of the reacting arrangement than of the unreactive ground-state complex.

Phenylalanine and leucine have hydrocarbon side chains and provide no conventional acid/base or hydrogen-bond donor/acceptor group here. They can position the enone, exclude unproductive solvent and select the attacked face through hydrophobic effect and steric hindrance. Those binding effects can assist catalysis but should not be drawn as an imaginary side-chain proton relay. The original schematic depicts the Glu water relay and the Gln/Arg oxyanion-stabilizing contacts.

Y91F implicates the Tyr hydroxyl, while E57Q implicates the Glu carboxylic-acid chemistry. Replacing tyrosine by phenylalanine retains an aromatic side chain but removes its hydroxyl, supporting a role for Tyr91 as a hydrogen bond donor that polarizes the steroid C3 carbonyl and stabilizes an enolate-like intermediate or transition state. Its hydroxyl could participate in a proton relay, but inactivity alone does not establish that it supplies the final proton.

Replacing a glutamate residue by glutamine approximately preserves side-chain size while replacing a titratable carboxylic acid by an amide. Loss of activity, combined with the acidic activity range, supports protonated Glu57 as a carbonyl-activating acid and possible proton donor. The two residues can cooperate to stabilize oxygen charge during reduction. The inhibitor-binding structure supports local contacts; it is not a direct picture of a testosterone transition state, and an N–H contact of the aza inhibitor need not occur in testosterone.

To distinguish these chemical roles from indirect effects, compare mutant and wild-type abundance, membrane insertion and folding, cofactor binding, and steroid binding. Measure initial rates with matched enzyme amounts and include no-enzyme and inhibitor-treated wild-type controls. If binding survives but turnover fails, the evidence for a catalytic defect is stronger. The published study combines structure, mutagenesis and modeled testosterone placement, so the proposed residue roles should be distinguished from direct observation of proton trajectories.^[ref]

catalysis requires a protonated group, consistent with a general-acid contribution. Loss of its proton as pH rises would remove carbonyl activation or the proton needed to finish the conjugate reduction. A glutamate residue with a shifted active-site $p{K}_a$ is a plausible candidate, but a pH cutoff alone neither identifies the residue nor measures a precise $p{K}_a$. Irreversible unfolding or changes in substrate binding must also be excluded by reversible pH jumps, protein-integrity checks and initial-rate measurements.

For the mechanistic notation below, the reduced cofactor is written NADPH, the established cofactor of human steroid 5α-reductase. The original PDF explicitly labels it NADH; its abbreviated N-substituent does not display the phosphate distinction. NADH and NADPH have the same reactive reduced nicotinamide ring, so the local hydride-transfer arrows are unchanged, although their biochemical specificity is different.^[ref]