1-Aminocyclopropanephosphonate: time-dependent inactivation of 1-aminocyclopropanecarboxylate deaminase and Bacillus stearothermophilus alanine racemase by slow dissociation behavior.
1-Aminocyclopropanephosphonate (ACPP) was synthesized, and its effects 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 less than 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 (Formula: see text) mechanism had an association rate constant (k1) of 100 M-1 s-1, a value 10(6)-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 k1 calculated in the inhibition studies.(ABSTRACT TRUNCATED AT 250 WORDS)
D000411:Alanine Racemase / Q000037:antagonists & inhibitors*; D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors*; D019755:Carbon-Carbon Lyases; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors*; D008433:Mathematics; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D011485:Protein Binding; D013053:Spectrophotometry
D009943:Organophosphorus Compounds; C053848:1-aminocyclopropanephosphonate; C033634:1-aminocyclopropane-1-carboxylate deaminase; D008190:Lyases; D019755:Carbon-Carbon Lyases; D000591:Amino Acid Isomerases; D000411:Alanine Racemase