Acetohydroxamic Acid - A Competitive Inhibitor of Urease from Soybean “Glycine max”

The acetohydroxamic acid (AHA), a potent inhibitor of urease, inhibits soybean urease competitively and reversibly. The I 50 and K i value for AHA were 900 M and 0.053 mM, respectively at pH 7.0, 37 °C. The variation in pH over the pH 6 9 affected K i and therefore binding of AHA in the active site. The affinity of AHA for the active site decreases with lowering of pH (below the pK a value of AHA i.e. 8.7). This behaviour is consistent with the deprotonated AHA acting as a nucleophile or the inhibitory species. The time-dependent inhibition studies were performed at two different concentrations of AHA and the biphasic kinetics was revealed with almost equal amplitudes (50% each) for fast and slow phases. The values of rate constants were 0.1642 ± 0.0013 min -1 (fast phase); 0.0123±0.0012 min (slow phase) at 0.10 mM AHA and 0.2379±0.0017 min (fast phase); 0.0153±0.0010 min (slow phase) at 0.15 mM AHA. These studies established the asymmetric nature of active sites, half being more reactive for AHA than the other half. The spectral studies showed a change in absorbance at the  max 414 nm, when urease was incubated with AHA, which was consistent with AHA binding to Ni of active site.