Michael N. Harris, Craig M. Bertolucci, and Li-June Ming*
Department of Chemistry and Institute for Biomolecular Science
University of South Florida
4202 Fowler Avenue
Tampa, FL 33620-5250
USA

Abstract
A phosphate was proposed to be a bridging ligand in the structure 1xjo of Streptomyces di-zinc aminopeptidase (sAP), which prompted further phosphate binding study.  Phosphate inhibits sAP and its Co²⁺-substituted derivatives (ZnZn-sAP, CoZn-sAP, and CoCo-sAP) in a noncompetitive manner from pH 6.0 to 9.0, with strongest inhibition observed at lower pHs (Ki = 0.6, 8.2, and 9.1 mM, respectively, at pH 6.0) which indicates that phosphate may not compete with substrate binding to the dinuclear active site.  The 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 may not be affected by the metal centers of different Lewis acidities.  Modification of ZnZn-sAP and CoCo-sAP with the arginine-specific reagent phenylglyoxal shows approximately a 10-fold increase in Ki and ~4–8-fold increase in Km, along with a significant decrease in kcat in both cases.  Furthermore, phosphate and the transition-state inhibitor 1-aminobutyl phosphonate can protect arginine from modification, strongly suggesting the involvement of arginines, such as Arg202 close to the active site, in phosphate binding and stabilization of the transition state.  Paramagnetic effect on ³¹P NMR relaxation of phosphate caused by Co²⁺ in CoZn-sAP has been measured, which reveals that only one phosphate is bound to sAP and the Co²⁺–³¹P distance in the range of 4.1–4.3 Å.  The ¹H NMR relaxation of the bulk water signal in the presence of CoCo-sAP remains unchanged as a function of phosphate concentration, further indicating that phosphate may not bind to the metal and displace any metal-bound water/hydroxide in the active site of sAP.  These results strongly suggest that the phosphate binding site is Arg202 and that this residue plays an important role in the action of sAP.