Past exam of the biochemistry and Molecular Biology course of the University of Cambridge / 2025 / Paper 2 / Section B / 11

The catalytic serine residue supplies a nucleophile: its side-chain oxygen attacks the carbonyl carbon of the beta-lactam ring. This generates a tetrahedral intermediate, followed by opening of the ring and formation of a covalent acyl-enzyme intermediate. Subsequent attack by activated water hydrolyzes that intermediate and regenerates the enzyme. The serine residue performs covalent catalysis and is regenerated, rather than being consumed as a reagent.

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]

Bond c, the carbonyl-carbon–nitrogen bond within the four-membered beta-lactam ring, is cleaved. Hydrolysis opens the cyclic amide; it does not cut the side-chain amide or a carbon–carbon bond. The resulting open-ring product no longer has the intact beta-lactam pharmacophore required for its usual antibacterial action.

The compound imidazole resembles the metal-coordinating part of the histidine side chain and competes for binding to the nickel sites in immobilized metal affinity chromatography. At the lower concentration in the wash, it helps displace weakly or non-specifically bound proteins while the multiple contacts of the histidine tag retain the desired enzyme. At the higher elution concentration, competition is strong enough to displace the tagged enzyme. Low imidazole improves washing; high imidazole elutes the target. Remove or match the elution-buffer components before interpreting downstream assays, and verify purity independently of total protein concentration.

The dilution in the Bradford assay is $1000/5=200$. Read the fitted line rather than assuming it passes through the origin: the two absorbances correspond approximately to $4.3$ and $15\mu \mathrm{g}\mathrm{m}{\mathrm{L}}^{-1}$ in the assay mixture. ThereforeMultiplying by the $0.500\mathrm{mL}$ eluate volume gives about $0.43\mathrm{mg}$ of TEM-1 and $1.5\mathrm{mg}$ of TEM-52. Only TEM-52 meets the requirement.

Assuming the same recovery per culture volume, the required volumes areUse approximately $120\mathrm{mL}$ or more for TEM-1; the existing $50\mathrm{mL}$ is sufficient for TEM-52. Rounding the former up to $125\mathrm{mL}$ is sensible given graphical precision. Reading the lower standard as $4$–$4.5\mu \mathrm{g}\mathrm{m}{\mathrm{L}}^{-1}$ gives a corresponding requirement of roughly $110$–$125\mathrm{mL}$, not an exact integer volume. These estimates assume comparable dye response to bovine serum albumin, suitable buffer blanks and that recovered protein is the purified target; contaminants would inflate the inferred target yield.

For the Hanes-Woolf plot, the slope is $1/V_{\max }$, the vertical intercept is $K_m/V_{\max }$, and the horizontal intercept is $-K_m$. The lines have approximately equal slopes, $0.50\min \mu {\mathrm{M}}^{-1}$. The dashed TEM-1 line intercepts the vertical axis at about $13\min$, while the dotted TEM-52 line intercepts it at about $2.5\min$. ThusThe negative-axis intercepts independently give approximately $-26$ and $-5\mu \mathrm{M}$. Allow normal graph-reading uncertainty, especially at the smaller intercept. The limiting rates are similar; the Michaelis constant is substantially lower for TEM-52. A Michaelis constant is not generally a binding dissociation constant, and calculating a turnover number additionally requires the concentration of active enzyme.

The displayed separation between the two entrance landmarks increases from approximately $6.2$ to $7.7\text{A˚}$ in TEM-52, indicating a wider opening. The Gly238-to-Ser substitution can alter backbone packing and loop geometry; its effect is not simply the steric size of the new side chain. Increased accessibility or altered productive positioning could increase the effective association rate for penicillin G, producing a lower apparent Michaelis constant without changing the limiting catalytic step. In a simple Michaelis-Menten kinetics scheme, $K_m=(k_{-1}+k_{\mathrm{cat}})/k_1$, so increasing $k_1$ can lower $K_m$.

The structural change is consistent with improved access and the lower measured $K_m$, while similar $V_{\max }$ suggests similar limiting turnover under these conditions. It does not uniquely establish the mechanism or prove tighter equilibrium binding. TEM-52 contains additional substitutions, so a matched single-substitution series would be needed to isolate the contribution of residue 238. Structural work on TEM-52 independently supports the importance of remodeling access to its active site.^[ref]

The bulky substituents of ceftazidime can hinder productive entry into the more restricted TEM-1 active site. A wider entrance in TEM-52 could accommodate this substrate and allow its beta-lactam ring to be hydrolyzed. The prediction is reduced susceptibility of TEM-52-expressing bacteria compared with matched TEM-1-expressing bacteria. This is a mechanistic prediction, not a quantitative conclusion from the penicillin G data: entry alone does not guarantee efficient catalytic chemistry for a different substrate. Test ceftazidime hydrolysis directly and measure its minimum inhibitory concentration in an otherwise matched bacterial background, controlling enzyme expression, growth, drug entry and other resistance mechanisms.