Paramagnetic Cobalt(II) as a Probe for Kinetic and NMR Relaxation Studies of Phosphate Binding and the Catalytic Mechanism of Streptomyces Dinuclear Aminopeptidase

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 Co 2 + -substituted derivatives in a noncompetitive manner from pH 6.0 to 9.0, with strongest inhibition observed at lower pHs (K i = 0.6, 8.2, and 9.1 mM for ZnZn-, CoCo-, and CoZn-sAP, respectively, at pH 6.0), which indicates that phosphate does not compete with substrate binding to the dinuclear active site and that monobasic phosphate has a higher binding affinity. The inhibition K i -pH profiles for phosphate inhibition of both the native and the Co 2 + -substituted derivatives reveal a similar plea around 7.0, reflecting that phosphate binding is not affected by the metal centers of different Lewis acidities. Modification of ZnZn- and CoCo-sAP with the arginine-specific reagent phenylglyoxal reveals a significant weakening in phosphate and substrate binding by showing approximately a 10-fold increase in the dissociation constant K for phosphate binding and ∼4-8-fold increase in K m . The catalysis is also influenced by the modification as reflected by a significant decrease in k c a t 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 transition state. The effect on 3 1 P NMR relaxation of phosphate caused by the paramagnetic metal center in Co 2 + -substituted derivatives of sAP has been measured, which reveals that only one phosphate is bound to sAP with the Co 2 + - 3 1 P distance in the range of 4.1-4.31. The 1 H NMR relaxation of the bulk water signal in the CoCo-sAP sample remains unchanged in the presence of phosphate, further indicating that phosphate may not bind to the active-site metals to displace any metal-bound water/hydroxide. These results strongly support that the phosphate binding site is Arg202 and that this residue plays an important role in the action of sAP.