| Abstract
| - Phosphate was proposed to be a bridging ligand in the structure of Streptomyces dizinc aminopeptidase (1xjo.pdb). However, phosphate inhibits sAP and its Co2+-substituted derivatives in a noncompetitive manner at pH 6.0−9.0, indicating that phosphate does not compete with substrate binding to the dinuclear active site. Along with a chemical modification and nuclear magnetic resonance experiments, Arg202 near the active site is proposed to be involved in phosphate binding and in stabilizing the transition state.
- Phosphate was proposed to be a bridging ligand in the structure 1xjo.pdb of Streptomyces dizinc aminopeptidase(sAP), which prompted further studies of phosphate binding to this enzyme. Phosphate inhibits sAP and its Co2+-substituted derivatives in a noncompetitive manner from pH 6.0 to 9.0, with strongest inhibition observed at lowerpHs (Ki = 0.6, 8.2, and 9.1 mM for ZnZn-, CoCo-, and CoZn-sAP, respectively, at pH 6.0), which indicates thatphosphate does not compete with substrate binding to the dinuclear active site and that monobasic phosphate hasa higher binding affinity. The inhibition Ki−pH profiles for phosphate inhibition of both the native and the Co2+-substituted derivatives reveal a similar pKa around 7.0, reflecting that phosphate binding is not affected by themetal centers of different Lewis acidities. Modification of ZnZn- and CoCo-sAP with the arginine-specific reagentphenylglyoxal reveals a significant weakening in phosphate and substrate binding by showing approximately a10-fold increase in the dissociation constant Ki for phosphate binding and ∼4−8-fold increase in Km. The catalysisis also influenced by the modification as reflected by a significant decrease in kcat in both cases. Furthermore,phosphate and the transition-state inhibitor 1-aminobutyl phosphonate can protect arginine from the modification,strongly suggesting that Arg202 near the active site is involved in phosphate binding and in stabilizing the transitionstate. The effect on 31P NMR relaxation of phosphate caused by the paramagnetic metal center in Co2+-substitutedderivatives of sAP has been measured, which reveals that only one phosphate is bound to sAP with the Co2+−31Pdistance in the range of 4.1−4.3 Å. The 1H NMR relaxation of the bulk water signal in the CoCo-sAP sampleremains unchanged in the presence of phosphate, further indicating that phosphate may not bind to the active-sitemetals to displace any metal-bound water/hydroxide. These results strongly support that the phosphate bindingsite is Arg202 and that this residue plays an important role in the action of sAP.
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