Past exam of the chemistry B course of the University of Cambridge / 2025 / Paper 2 / B10 / b

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]

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]

Hydride is delivered to steroid C5 and a proton to C4; the C3 ketone is retained. Use the conjugated fragment $\mathrm{O}=C3-C4=C5$. Carbonyl hydrogen bonds from Tyr91–OH and protonated Glu57 lower the barrier to conjugate reduction. Draw a curved arrow from the nicotinamide C4–H bond of NADPH to steroid C5, shift the C4=C5 pi pair toward C3–C4, and shift C3=O pi electrons onto oxygen. The nicotinamide ring rearomatizes to NADP+; the steroid becomes a bound enolate, or its protonated enol equivalent. electron shifts across the nicotinamide ring must accompany C–H bond cleavage rather than leaving an unexplained neutral carbon radical.

Next, reform C3=O and protonate steroid C4 from an appropriately positioned acid, directly or through an enol/proton-relay route. This regenerates the catalytic acid after solvent reprotonation. The enzyme fixes the hydride-accepting face so that the new C5–H has the 5α configuration, giving dihydrotestosterone; delivery to the opposite face would give a different ring-junction configuration. This is not simple 1,2 reduction of the C3 carbonyl to an alcohol. The net local bookkeeping is one hydride plus one proton across C4=C5, with two electrons supplied by NADPH. The original reactive-fragment diagram shows the enolate-forming arrows and the separate protonation step.

finasteride is reduced at C1, leaving a C2 enolate that attacks nicotinamide C4 to make the covalent C–C adduct. In the inhibitor's unsaturated lactam, the reactive chain is $\mathrm{O}=C3-C2=C1$, with N at steroid position 4. Activate the C3 carbonyl by hydrogen bonding; draw the reduced cofactor's C4–H bond toward inhibitor C1, the C1=C2 pi pair toward C2–C3, and the C3=O pi pair toward oxygen. This gives NADP+ next to the C2-centered resonance form of the inhibitor enolate.

Instead of completing reduction by protonating C2, move the C2 lone pair toward the electrophilic C4 of the oxidized nicotinamide ring. Shift the pyridinium pi system toward its positively charged N, and reform the inhibitor carbonyl if using the oxygen-centered enolate representation. The result is the C2(inhibitor)–C4(nicotinamide) bond and a neutral dihydronicotinamide ring. Numbering the two molecules separately prevents confusing cofactor C4 with the inhibitor's N4. The lactam N–H remains present and can help position the inhibitor through hydrogen bonding; it does not form a covalent bond to the protein.

This is mechanism-based inhibition: enzyme chemistry generates the species that traps its own oxidized cofactor. Isolation of a cofactor–inhibitor adduct after protein denaturation, rather than persistent attachment to a denatured protein, distinguishes it from covalent modification of an amino acid side chain. The original mechanistic study combined radiolabel recovery, purification and spectroscopy to establish the adduct; its slow breakdown also cautions against treating “irreversible” as an absolute statement for every timescale.^[ref]