Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters

Abstract

Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C. A Brønsted plot of log (kcat/Km) (M-1 s-1) vs. the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.

Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase.

“…For each mutant, the kcat values for two different substrates are very similar, showing the same lack of dependence on the substrate leaving group that has been so thoroughly documented for the wild-type enzyme (4). For each enzyme, though, there is a marked increase in the Km value for phenyl phosphate relative to that for 4-nitrophenyl phosphate, in contrast to the reported (4) measurements for the wild-type enzyme. Based on the Michaelis-Menten parameters at pH 8 and 250C with two substrates of widely different pK values, a Bronsted plot of log (kcat/Km) vs. the pK of the leaving group would give a slope (PLG) of -0.36 for both mutants.…”

“…One of the striking features of alkaline phosphatase is the lack of sensitivity ofthe kcat/Km values for mnonophosphate ester hydrolysis toward the leaving group of the substrate. It has been proposed that the very small change in effective charge on the leaving group's oxygen in the course of the reaction is consistent with substantial electrophilic interaction of groups in the enzyme with this atom (4). The role ofthis electrophile could be filled by either a zinc ion or by the side chain of arginine-166.…”

Enzymatische Acyl‐ und Phosphoryltransferreaktionen unter Beteiligung von zwei Metallionen

“…Adenosylmethionin-SynthetaseDie (S)-Adenosylmethionin-Synthetase (AMS) katalysiert die Bildung der Sulfonverbindung (S)-Adenosylmethionin (AdoMet), die als bedeutendes Methylierungsreagens in biologischen Systemen fungiert, aus L-Methionin und ATP (Abb 25…”

Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis byPseudomonas aeruginosaArylsulfatase

“…The mechanism of transition state (TS) stabilization during enzymecatalyzed substrate hydrolysis of one member of this superfamily, Escherichia coli alkaline phosphatase (EcAP), has been subject of a large number of in-depth studies involving the experimental tools of linear free energy relationships (LFERs), kinetic isotope effects (KIEs), mutant studies, and structural analysis by X-ray crystallography. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] In addition computational simulations have been employed to pin-point transition state interactions. 26,27,[37][38][39][40][41] Sulfatase members of the AP superfamily have been studied less extensively.…”

“…of human arylsulfatase A, 48 choline sulfatase, 47 and the closely related phosphonate monoester hydrolase 49 ) have suggested that many of these conserved residues are indeed involved in the catalytic pathway in the enzyme active site. LFERs and KIEs have been measured for a number of phosphatases, [20][21][22][23]35,[50][51][52][53][54][55][56][57][58] but only one such study is available for sulfatases. 59 In addition to the family relationship, members of the AP superfamily are also typically catalytically promiscuous, [60][61][62] i.e.…”