MCAT Practice Test 2

Inhibitors of a bacterial serine-pathway enzyme

¹Many soil bacteria synthesize serine through a three-step pathway whose first committed step is catalyzed by PsdA, an ${\text{NAD}}^{+}$-dependent dehydrogenase that oxidizes 3-phosphoglycerate (3PG). PsdA is a homotetramer, and bacteria lacking it cannot grow in serine-poor soil. A research group therefore screened small molecules for inhibitors of purified PsdA that might serve as leads for selective antibacterial agents.

²In Experiment 1, initial rates of NADH formation were measured spectrophotometrically at 25 °C with a fixed concentration of PsdA, saturating ${\text{NAD}}^{+}$ and 3PG concentrations from 0 to 60 mM. Assays contained no inhibitor, 10 µM compound C1 or 10 µM compound C2 (Figure 1). Each point is the mean of three assays, and standard deviations were less than 5% of the mean. Rates measured at 500 mM 3PG (not plotted) were 99, 98 and 50 µmol/min per mg protein for no inhibitor, C1 and C2, respectively.

³For an inhibitor that binds only the free enzyme, the apparent Michaelis constant is $K_m^{app}=K_m(1+[\text{I}]/K_i)$, where $[\text{I}]$ is the inhibitor concentration and $K_i$ is the dissociation constant of the enzyme–inhibitor complex.

⁴In Experiment 2, a third compound, C3, was tested at 10 µM. Kinetic constants obtained from a double-reciprocal (Lineweaver–Burk) plot of the C3 data are listed in Table 1 with the no-inhibitor values.

⁵In Experiment 3, the Experiment 1 assay was repeated with 1 mM serine, the end product of the pathway, and no inhibitor. The plot of rate versus [3PG] became sigmoidal, with half-maximal rate at 20 mM 3PG and a Hill coefficient of 2.6, compared with 1.0 without serine. The rate at 500 mM 3PG was unchanged by serine.