1-Aminocyclopropanephosphonate: time-dependent inactivation of 1-aminocyclopropanecarboxylate deaminase and Bacillus stearothermophilus alanine racemase by slow dissociation behavior

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Abstract
1-Aminocyclopropanephosphonate (ACPP) was synthesized, and its effect on the pyridoxal 5''-phosphate linked enzymes 1-aminocyclopropanecarboxylate (ACPC) deaminase from Pseudomonas sp. ACPC and alanine racemase from Bacillus stearothermophilus were studied. ACPP was found to be a potent inhibitor of both enzymes with Km/Ki ratios of 500 and 2000, respectively. Inhibition for both enzymes was characterized by slow-binding (second-order rate constants < 150 M-1 s-1) slow-dissociating behavior. Analysis of the pre-steady-state kinetics revealed a kinetically detectable intermediate E-I complex in the inhibition mechanism for the racemase but not for the deaminase. The one-step deaminase inhibition mechanism had an association rate constant (ki) of 100 M-1 s-1, a value 106-fold slower than diffusion, suggesting either a slow alignment of the inhibitor at the enzyme active site, or more likely, the same mechanism as followed by racemase but with an E-I to E-I* conversion rate (k3) that is sufficiently fast on the steady-state time scale so as to hinder detection of the initial weakly associated E-I intermediate. The E to E-I transition for the deaminase was further monitored by ultraviolet-visible and circular dichroism (CD) spectroscopies and found to exhibit a time-dependent shift in the visible absorption spectrum .lambda.max from 418 nm for the native enzyme to 333 nm at steady state, again consistent with a rapid E to E-I and slow E-I to E-I* behavior. A rate constant for the absorbance shift of 150 M-1 s-1 was consistent with the ki calculated in the inhibition studies. The CD spectra showed a similar absorbance shift along with a change in the optical rotation direction. Sodium borohydride failed to reduce the deaminase-ACPP complex, suggesting that the 333-nm absorbance might arise from formation of a stable hemiaminal.