Rotational resonance determination of the structure of an enzyme-inhibitor complex: phosphorylation of an (aminoalkyl)phosphinate inhibitor of D-alanyl-D-alanine ligase by ATP

Abstract

We have used a newly developed solid-state NMR method, rotational resonance, to establish the structure of an inhibited complex formed upon reaction of D-alanyl-D-alanine ligase, ATP, and the aminoalkyl dipeptide analogue [1(S)-aminoethyl][2-carboxy-2(R)-methyl-1-ethyl]phosphinic acid (Ib). Analogue Ib was determined to be an ATP-dependent, slow-binding inhibitor of the D-Ala-D-Ala ligase from Salmonella typhimurium, with an enzyme-inhibitor half-life of 17 days at 37.degree. C. The inhibited complex shows a 31P NMR spectrum which is very different from that which would arise from a mixture of the free inhibitor and ATP. Four well-resolved lines were observed: two (at -8 and -14 ppm) are assignable as the phosphates of ADP, the third is assignable to an inhibitor resonance (at 53 ppm) that shifts by approximately 19 ppm on binding, and the fourth is assignable to a resonance (at -3 ppm) due to a polyphosphate or phosphate ester moiety. At rotational resonance the spectrum shows evidence for strong dipolar couplings between the phosphinate phosphorus and a phosphate ester species. The dipolar coupling between the phosphorus signals at 53 and -3 ppm was measured at rotational resonance by use of numerical stimulations of both the line shape of the signal and the profile of magnetization transfer between the two sites. The measured coupling, 1.0 .+-. 0.2 kHz, indicates that the two species are bridged in a P-O-P linkage, with a P-P through-space distance of 2.7 .+-. 0.2 .ANG.. This proves that the mechanism of inactivation involves phosphorylation of the enzyme-bound inhibitor by ATP to form a phosphoryl-phosphinate adduct.