Hydrogen-bonding interactions in the active site of a low molecular weight protein-tyrosine phosphatase

Abstract

Molecular dynamics simulation of the Michaelis complex, phospho-enzyme intermediate, and the wild-type and C12S mutant have been carried out to examine hydrogen-bonding interactions in the active site of the bovine low molecular weight protein-tyrosine phosphatase (BPTP). It was found that the S γ atom of the nucleophilic residue Cys-12 is ideally located at a position opposite from the phenylphosphate dianion for an inline nucleophilic substitution reaction. In addition, electrostatic and hydrogen-bonding interactions from the backbone amide groups of the phosphate-binding loop strongly stabilize the thiolate anion, making Cys-12 ionized in the active site. In the phospho-enzyme intermediate, three water molecules are found to form strong hydrogen bonds with the phosphate group. In addition, another water molecule can be identified to form bridging hydrogen bonds between the phosphate group and Asp-129, which may act as the nucleophile in the subsequent phosphate hydrolysis reaction, with Asp-129 serving as a general base. The structural difference at the active site between the wild-type and C12S mutant has been examined. It was found that the alkoxide anion is significantly shifted toward one side of the phosphate binding loop, away from the optimal position enjoyed by the thiolate anion of the wild-type enzyme in an S N 2 process. This, coupled with the high pK a value of an alcoholic residue, makes the C12S mutant catalytically inactive. These molecular dynamics simulations provided details of hydrogen bonding interactions in the active site of BPTP, and a structural basis for further studies using combined quantum mechanical and molecular mechanical potential to model the entire dephosphorylation reaction by BPTP.

Free-energy profiles for catalysis by dual-specificity phosphatases

“…Although the C-O and O-P bond lengths in the C124S mutant are shorter than the corresponding C-S and S-P bond lengths in the WT VHR, dephosphorylation would occur if Ser 124 were ionized. Hence, the phosphate group has enough mobility in the P-loop to be attacked by an ionized Ser 124 nucleophile, even if O γ is not optimally located [22,23].…”

“…including structural alterations in the mutant [21][22][23], increased distance between nucleophile and substrate [22] and nucleophile protonation [23].…”

Mechanisms and Free Energies of Enzymatic Reactions

“…In fact, this system provides an excellent example, illustrating the significance of correlating structural information from computation and X-ray crystallography with enzyme activity to understand catalysis. 193,194 The phosphate binding site of PTP is characterized by the signature loop with a sequence of CXX-NXXR(S/T), where X can be any residue. The nucleophile Cys12 is stabilized by hydrogen bonding interactions from the side chains of Asn15 and Ser19 and the amide group of Ser19, making it a thiolate ion by lowering its pK a 195 and positioning it in the center of the binding loop perfectly suited for the in-line nucleophilic attack (Figure 3).…”

“…94,276 Correlation of catalysis with the change in average hydrogen bond distance in the enzyme has been analyzed for other systems, including the PTP phosphate hydrolysis reaction, and its origin in that case was attributed to the Walden inversion mechanism in the nucleophilic substitution reaction. 193,194 …”

Intermolecular hydrogen bonding of N-methylformamide in aqueous environment: A theoretical study

“…It has been found that water prefers to form hydrogen bonds with oxygen atoms of peptide bonds in proteins [2,3]. Studies of the interactions between water and other molecules are important to understand more fully the role played by water in the biochemical behavior of the molecules [4][5][6].…”