Trapping and visualization of a covalent enzyme–phosphate intermediate

Murphy, Jennifer E.; Stec, Boguslaw; Ma, Lan; Kantrowitz, Evan R.

doi:10.1038/nsb0897-618

Cited by 55 publications

(45 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Evidence in support of this model comes from x-ray structures with covalently and noncovalently bound phosphate and with a bound pentacoordinate vanadate transition state analog. 19,20,55 Functional data support an important energetic interaction of this oxygen atom with the active site bimetallocluster. R166S AP exhibits a remarkable correlation between the amount of charge on this oxygen atom and the observed rate enhancement.…”

Section: Analysis Of Interactions With the Nonbridging Oxygen Atomsmentioning

confidence: 99%

Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction

et al. 2007

View full text Add to dashboard Cite

The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here we use kinetic isotope effects (KIEs) to explore the origin of the 10 9 -fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18 O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of pnitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are 'loose' and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn 2+ ion. Furthermore, the KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn 2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.

show abstract

Section: Analysis Of Interactions With the Nonbridging Oxygen Atomsmentioning

confidence: 99%

Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction

et al. 2007

View full text Add to dashboard Cite

show abstract

“…2 R166 appears to interact with the nonbridging phosphoryl oxygen atoms of a phosphate monoester (65) and could stimulate the phosphomonoesterase reaction by helping to bind and position the substrate, by stabilizing charge rearrangement in the transition state, and/ or by desolvating the phosphoryl group (49,(65)(66)(67). This interaction does not contribute to the reaction of phosphate diesters (Tables 2 and 3), presumably because the introduction of steric bulk upon esterification of an oxygen of the transferred phosphoryl group prevents this interaction with the phosphate diester substrate.…”

Section: Discussionmentioning

confidence: 99%

Functional Interrelationships in the Alkaline Phosphatase Superfamily: Phosphodiesterase Activity of Escherichia coli Alkaline Phosphatase

2001

View full text Add to dashboard Cite

Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn 2+ ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono-and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10 11 -fold relative to the rate of the uncatalyzed reaction [(k cat / K m )/k w ]. Although this rate enhancement is substantial, it is at least 10 6 -fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosphatases, and phosphodiesterases within the evolutionarily related AP superfamily. The catalytic promiscuity of AP could have facilitated divergent evolution via gene duplication by providing a selective advantage upon which natural selection could have acted.Two divalent metal ions are a common active site feature found in a number of evolutionarily distinct classes of enzymes, including enzymes that catalyze the hydrolysis of phosphate monoesters, diesters, and triesters (for reviews, see refs 1 and 2). The structural similarity shared by these active sites raises the possibility that a single active site with two divalent metal ions might be able to catalyze these different classes of reactions. However, the partial negative charge on a phosphoryl oxygen atom of the phosphate monoester dianion is lost upon conversion to a diester monoanion, and the steric bulk of the added esterifying group of the diester could result in steric clashes with...

show abstract

“…46 In the presence of P i , the enzyme exists in a pH-dependent equilibrium between the E ž P i and E-P forms. For a wildtype enzyme, the equilibrium significantly favors the E-P form at pH 5.5.…”

Section: X-ray Structure Of the Covalent Enzyme-phosphate Complexmentioning

confidence: 99%

E. coli Alkaline Phosphatase

Kantrowitz

2004

Handbook of Metalloproteins

View full text Add to dashboard Cite

Escherichia coli alkaline phosphatase (AP) is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphomonoesters. Because of the extensive functional and structural studies available on the AP from E. coli , this enzyme has become the model for all APs. The two active sites of dimeric AP each contain binding sites for two Zn 2+ and one Mg 2+ . These metals are critical for dimer formation and catalytic activity. The reaction proceeds via a phosphoseryl intermediate formed by the attack of the hydroxyl oxygen of Ser102 on the phosphorus of the substrate. The substrate is stabilized in the active site by binding to Zn 2+ . In the second step of the double in‐line displacement mechanism, a hydroxyl bound to Zn 2+ hydrolyzes the phosphoseryl intermediate. Under basic conditions, the slow step in the mechanism is the release of phosphate from the noncovalent enzyme–phosphate complex. X‐ray structures of the free enzyme, the phosphoseryl intermediate, an enzyme–vanadate complex that mimics the transition state, and the enzyme–phosphate noncovalent complex have been used to delineate molecular details of the catalytic mechanism. Sequence alignments of APs from various species have been used to provide explanations for the enhanced activity of the mammalian APs as compared to the bacterial APs and why certain APs utilize Co 2+ in place of Zn 2+ . The molecular details of phosphate hydrolysis ascertained from the study of AP has applications to other enzymes that are involved in phosphate ester hydrolysis.

show abstract

Trapping and visualization of a covalent enzyme–phosphate intermediate

Cited by 55 publications

References 29 publications

Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction

Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction

Functional Interrelationships in the Alkaline Phosphatase Superfamily: Phosphodiesterase Activity of Escherichia coli Alkaline Phosphatase

E. coli Alkaline Phosphatase

Contact Info

Product

Resources

About