ATP-dependent inactivation and slow binding inhibition of salmonella typhimurium D-alanine: D-alanine ligase (ADP) by aminoalkylphosphinate and aminophosphonate analogs of D-alanine (original) (raw)

In Salmonella typhimurium, D-a1anine:D-alanine ligase (ADP) (EC 6.3.2.4) is the second enzyme in the three enzyme palanine branch pathway of peptidoglycan biosynthesis. The interaction of this enzyme with a possible transition-state analogue, the (aminoalky1)phosphinate D-3-[ (l-aminoethyl)phosphinyl]-2heptylpropionic acid [Parsons et al. (1987) Abstracts of Papers, 193rd National Meeting of the American Chemical Society, Denver, CO, MEDI 63, American Chemical Society, Washington, DC] , has been studied. This compound is a potent active site directed inhibitor and is competitive with D-alanine (Ki = 1.2 pM); it exhibits time-dependent inhibition in the presence of ATP. Kinetic analysis revealed a rapid onset of steady-state inhibition (k,, = 1.35 X lo4 M-' s-' ) followed by slow dissociation of inhibitory complex(es) with a half-life of 8.2 h. The inhibitory complex was shown to consist of E-.I--ATP in equilibrium with E--I, Pi, and ADP. Similar time-dependent inhibition was also observed with D-( 1-aminoethy1)phosphonic acid (D-Ala-P) (Ki = 0.5 mM; k,, = 27 M-' s-l; t l I 2 for regain = 1.73 min) but not with D-(1-amino-ethy1)phosphinic acid, which behaved as a simple competitive inhibitor (Ki = 0.4 mM). The mechanism of inhibition is discussed in the light of the precedents of glutamine synthase inhibition by methionine sulfoximine and phosphinothricin.

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