Step 2 Phosphoenzyme hydrolysis

Figure 4. Simplified scheme for the reaction cycle in Ca2+ pumps. The pumps may adopt two major conformations E, and E2. The E, conformation shows high affinity for two Ca2+ (SERCA pumps) or one Ca2+ (PMCA pumps) on the cis side. Ca2+ binding greatly enhances the pumps ATPase activity, leading to the rapid formation of the h igh-energy phosphorylated intermediate E, P and occlusion (occ) of the transported Ca2+ ion(s). Ca2+ translocation across the membrane presumably occurs concomitantly with the release of energy stored as conformational constraint during the transition from the E, P to the low-energy E2-P conformation. Ca2+ affinity on the trans side is low and Ca2+ is therefore released. This is followed by hydrolysis of the phosphoenzyme and a poorly understood rearrangement step(s) from the E2 to the E, conformation.

The mechanism of the PTP hydrolysis reaction has two steps. First, phosphate is transferred from tyrosine to the cysteine residue of the P-loop, which generates a phosphoenzyme intermediate with concomitant release of tyrosine. This process is followed by hydrolysis of the phosphoenzyme to free enzyme and inorganic phosphate. Two active site residues are of primary importance during the catalytic cycle the nucleophilic cysteine of the P-loop and an aspartate on a nearby flexible loop, which serves as a general acid/base catalyst (Fig. 3). After attack of the cysteine on phosphotyrosine, tyrosine can be expelled as the protonated phenol after proton donation by the catalytic aspartic acid, which forms the phosphoenzyme intermediate and free tyrosine. The aspartate anion then deprotonates the hydrolytic water molecule that attacks phosphocysteine, which liberates inorganic phosphate (Fig. 4) (9). 

Consistent with the KIE results, LFER studies showed that /feat for the hydrolysis of aryl phosphomonoesters by native YopH exhibits almost no dependence on the basicity of the leaving group between pK 1 and 15. In contrast, a strong negative /3ig=—1.3 is found in reactions of mutants in which general acid catalysis is disabled. The fact that alcohols as well as water can dephosphorylate the phosphoenzyme intermediate was utilized to evaluate the transition state of this step. It was found that, for native Stpl and the YopH mutant Qfl46A, the / nuc 015, indicative of little nucleophilic participation and, presumably, a loose transition... 

Figure 17-7 Two alternative mechanisms utilized by phosphatases to carry out hydrolysis of phosphate esters. The phosphoenzyme intermediate mechanism utilizes an amino acid (represented as -X] as a nucleophile to attack the phosphate ester, transferring the phosphoryi group and producing a short-lived phosphoenzyme intermediate. In the second step, water serves as the nucleophile, hydrolyzing the phosphoenyzme intermediate and regenerating the enzyme. This mechanism is used by the tyrosine phosphatases (nucleophile = cysteine) and E. coli alkaline phosphatase (active site nucleophile = Ser 102). The metallophosphatases do not proceed by formation of a phosphoenzyme intermediate but rather carry out hydrolysis by direct transfer of the phosphoryi group to a metal-coordinated water molecule.

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