= Solution
\b[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 (biochemistry)> 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.<Structure of human steroid 5α-reductase 2 with the anti-androgen drug finasteride by Qingpin Xiao and others>[2020, Fig. 3a,c–e, structural contacts, docking and mutant activity comparison]{ref}
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