Mechanism of substrate dephosphorylation in lowMr protein tyrosine phosphatase

Abstract

Substrate dephosphorylation by the low molecular weight protein tyrosine phospha-tases proceeds via nucleophilic substitution at the phosphorous atom yielding a cysteinyl phosphate intermediate. However, several questions regarding the exact reaction mechanism remain unanswered. Starting from the crystal structure of the enzyme we study the energetics of this reaction, using the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two mechanisms corresponding to different protonation states of the reacting groups are examined along stepwise and concerted pathways. The activation barriers calculated relative to the enzyme-substrate complex are very similar for both monoanionic and dianionic substrates, but taking the substrate binding step into account shows that hydrolysis of monoanionic substrates is strongly favored by the enzyme, because a dianionic substrate will not bind when the reacting cysteine is ionized. The calculated activation barrier for dephosphorylation of monoanionic phenyl phosphate according to this novel mechanism is 14 kcal mol 1 , which is in good agreement with experimental data. Proteins 1999;36:370-379. 1999 Wiley-Liss, Inc.

Computational modeling of the rate limiting step in low molecular weight protein tyrosine phosphatase

“…In this work, we have studied the energetics of the second step of the reaction catalyzed by the low M r PTP (for review see [16,17]), using the empirical valence bond (EVB) method in combination with molecular dynamics (MD) and free energy perturbation (FEP) simulations [18,19]. The obtained reaction free energy pro¢le is combined with that of the ¢rst reaction step published by us elsewhere [20,21] and the calculated overall rate limiting barrier of 16 kcal/mol agrees well with the observed turn-over rate for the wild-type enzyme. An alternative reaction mechanism has been investigated for the mutant enzyme lacking the general acid/base (Asp-129 in low M r PTP) and the e¡ect of mutating Cys-17 is examined.…”

“…It is clear that the characteristic signature motif corresponding to the phosphate binding loop is the key catalytic factor in these enzymes. It appears to be optimized for stabilizing a double negative charge in the center of the loop (it was earlier shown that the enzyme cannot accommodate a total charge of 33 [21]). This explains both the e¤cient stabilization of the transition states and why sig-ni¢cant accumulation of the reaction intermediate does not occur.…”

“…1. We have shown in previous work [20,21] that with a negatively charged nucleophile, the substrate of low M r PTP is bound in its mono-anionic form, i.e. the reacting groups (Cys-12, Asp-129 and the phosphate moiety) have a total charge of 32.…”

“…) have been presented elsewhere[20,21]. x' 3^x ' 6 represent the alternative reaction pathway that may be used by mutants lacking the general acid Asp-129.…”

The catalytic mechanism of protein tyrosine phosphatases revisited

“…The improbability of binding a dianion, as opposed to a monoanion, in the active site carrying an ionised nucleophile was further shown by Kolmodin et al using binding free energy calculations [34]. They also reproduced the energetics of the whole reaction pathway for both the wild type and D128A mutant LMPTP using a monoanionic substrate [29].…”