Hydrolysis Phosphoenzyme

Acetate kinase is phosphorylated by acetyl phosphate and it has been shown that the phosphoenzyme can synthesise ATP from ADP, and acetyl phosphate from acetate. The mode of decomposition of carbamyl phosphate in aqueous solution is pH dependent and can proceed with either the production of ammonia and carbon dioxide (equation 1), or cyanate (equation 2). No cyanate could be detected during the hydrolysis... 

The rate of phosphoprotein formation in the presence of 5 mM CaCl2 was only slightly affected by mild photooxidation in the presence of Rose Bengal, but the hydrolysis of phosphoenzyme intermediate was inhibited sufficiently to account for the inhibition of ATP hydrolysis [359]. The extent of inhibition was similar whether the turnover of E P was followed after chelation of Ca with EGTA, or after the addition of large excess of unlabeled ATP. These observations point to the participation of functionally important histidine residues in the hydrolysis of phosphoprotein intermediate [359]. 

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

There is evidence (but not conclusive evidence) that the mechanisms involves direct phosphoryl transfer to water rather than the formation of a phosphoenzyme intermediate.293 This utilizes the fact that ATP also serves as a substrate to inorganic pyrophosphatase. Hydrolysis of the ATP analogue adenosine 5 -0-(3-thiotriphosphate) chirally labelled with I70 and I80 at the y-phosphate proceeds with inversion of configuration to give the chiral [170,180]-thiophosphate (Figure 13).293... 

Figure 22. Analysis of the rates of dephosphorylation of phosphoenzyme intermediates of wild-type and Pro312 mutants of the SR Ca2+-ATPase. Left panel Phosphorylation was performed with [y-32P]ATP in the presence of Ca2+. The buffer conditions were adjusted to obtain predominantly E,P in steady state as indicated by the complete disappearance of the phosphoenzyme upon addition of ADP. EGTA was added to terminate phosphorylation by chelation of Ca2+ and permit observation of the dephosphorylation of E,P through conversion to E2Pand hydrolysis of the latter intermediate. A rapid dephosphorylation is observed with the wild type, while in the mutants the dephosphorylation of E,P is inhibited. More than 80% of the phosphoenzyme remains five minutes after addition of EGTA in the Pro312- Ala mutant. Right panel Phosphorylation was performed with [32P]Pj by the backdoor reaction in the absence of Ca2+...

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). 

Protein phosphatases that are specific for phosphoserine/ phosphothreonine have a distinct reaction mechanism from tyrosine phosphatases. Protein serine phosphatases are transition metal-dependent, and the reaction mechanism does not involve a phosphoenzyme intermediate as in the case of PTPs. Crystal structures of multiple protein serine phosphatases have revealed how the enzymes catalyze hydrolysis of phosphoserine (14). 

Taken together, the data indicate direct phosphoryl transfer to a metal-bound water molecule without a phosphoenzyme intermediate. A Bronsted analysis found a value of (j of -0.3 for V/K,137 similar to the value for the uncatalyzed hydrolysis of phosphate monoester monoanions, which could be indicative of charge neutralization on the leaving group in the transition state via protonation. 

Biochemical alterations have been found in fragmented sarcoplasmic reticulum isolated from dystrophic human, mouse and chicken muscle. Alterations in calcium transport, ATP hydrolysis and phosphoenzyme formation have been reported. Some of these biochemical alterations in the dystrophic sarcoplasmic reticulum are suggested to be due to alterations of the lipid environment of these membranes it has been suggested that the cholesterol content of dystrophic sarcoplasmic reticulum is elevated [182-187]. 

In early studies the kinetics appeared to be sequential because of the convergence of double-reciprocal plots (72, 77). It was not entirely clear at the time that the phosphatase activity was an intrinsic part of the phosphotransferase activity, and the kinetics was not pursued further. Efforts to isolate an active phosphoenzyme failed, presumably due to its hydrolysis in the absence of a nucleoside 73, 77). The stereochemical analysis showing retention of configuration at phosphorus reopened the mechanistic question and stimulated further studies that... 

All phosphatases catalyze the same net reaction, the hydrolysis of a phosphate monoester to inorganic phosphate and the alcohol or phenol from the ester group. As already mentioned, despite the thermodynamic favorability of this reaction, the kinetic barrier is formidable. A number of the enzymes that catalyze this reaction have been characterized. Phosphatases vary in their preference for the charge state of the substrate (either the monoanion or the dianion), in the presence or absence of a metal center, and in the utilization of a phosphoenzyme intermediate versus direct attack by water. Even among metallophosphatases, there are variations in the means by which the dinuclear metal center participates in binding and catalysis. 

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... 

The last mediator of gastric secretion in the parietal cell is an H+,K+-ATPase (proton or acid pump) which is a member of the phosphorylating class of ion transport ATPases. Hydrolysis of ATP results in ion transport. This chemical reaction induces a conformational change in the protein that allows an electroneutral exchange of cytoplasmic H+ for K+. The pump is activated when associated with a potassium chloride pathway in the canalicular membrane which allows potassium chloride efflux into the extracytoplasmic space, and thus results in secretion of hydrochloric acid at the expense of ATP breakdown. The activity of the pump is determined by the access of K+ on this surface on the pump. In the absence of K+, the cycle stops at the level of the phosphoenzyme [137]. 

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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