Stereochemistry of phospho transfer catalyzed by bovine liver acid phosphatase.

Walden-Inversion-Enforced Transition-State Stabilization in a Protein Tyrosine Phosphatase

“…The temperature dependencies of rate constants from 4 to 23 °C are listed in Table for each of the individual chemical steps. As observed previously,8a burst kinetics was obeyed at each temperature with the rate-limiting step being the hydrolysis of the intermediate ( k 3 ), and the burst size corresponded to the stoichiometric concentration of BPTP. Activation parameters calculated from the data in Table are the following: Δ H ⧧ = 12.0 kcal/mol and Δ S ⧧ = −7.8 cal/(mol) K) for k 2 step; ΔH ⧧ = 13.7 kcal/mol and Δ S ‡ = −8.6 cal/(mol K) for k 3 step.…”

“…Initial coordinates of the enzyme−substrate complex are obtained by docking a tyrosine phosphate dianion residue into the active site region, with the position of the crystallographically determined phosphate ion as the anchoring point. BPTP has its maximum activity in the pH range of 4−8, according to which the state of all titratable residues is assigned. , Consequently, the two histidine residues (His66 and His72) are protonated in accord with experimental p K a values of 8.36 and 9.19, respectively. 26a,b There are two cysteine residues in the active site of BPTP, Cys12 and Cys17. The p K a of these two residues have been determined by titration with iodoacetate and iodoacetamide to be <4 for Cys12 and 9.1 for Cys17 26c…”

“…Pre-steady-state kinetic measurements of the BPTP-catalyzed hydrolysis of pNPP were conducted using a Hi-Tech SF-61 stopped-flow spectrophotometer (dead time, 2 ms) with an observation cell length of 1.0 cm. Fast reactions were studied at pH 7 by monitoring the increase in absorbance at 410 nm of the p -nitrophenolate product 8a. The buffer used was 50 mM 3,3-dimethylglutarate, 1 mM EDTA, I = 0.15 M adjusted by adding NaCl.…”

“…A number of crystal structures of protein tyrosine phosphatases have been determined, including the bovine PTPase (BPTP), the catalytic domain of human PTP1B, and the Yersinia PTPase ,, Each of the two steps involves a Walden inversion of configuration at the phosphorus center in the S N 2(P) process. The invariant Cys residue (e.g., Cys12 in BPTP, Cys215 in PTP1B, and Cys403 in the Yersinia PTPase) has been identified as the nucleophile in the initial step, and the invariant Arg at the end of the catalytic loop is necessary for both substrate binding and transition-state stabilization. − Because of the biological importance of phosphate hydrolysis, there have been numerous experimental investigations, and there is continuous interest in the understanding of the detailed catalytic mechanism for dephosphorylation reactions. − Early computational studies of phosphate hydrolysis are exemplified by gas-phase geometry optimizations using ab initio methods .…”

“…These studies constitute the basis for further investigations of this important reaction. The formation of a phosphocysteine intermediate in the PTPase-catalyzed reactions is particularly interesting, , since thiophosphate linkage in proteins has only been observed in bacterial thioredoxin and mannitol carrier-specific transport enzyme II its biological importance has only recently begun to be appreciated. ,,, Consequently, computational investigations of the reaction mechanism employing hybrid quantum mechanical and classical potentials are warranted to gain an understanding of phosphate thioester as an intermediate in phosphoryl transfer processes.…”