Alkaline phosphatase nucleophilicity

The two-step mechanism of phosphate ester hydrolysis by the (Znn)2-containing alkaline phosphatase (AP) (7) is thus somewhat mimicked by 24. The phosphoryl intermediate 25 is generated by nucleophilic attack of the alkoxide moiety in 24b at BNP" and is hydrolyzed by the intramolecular Zn11—OH" species in 25b. Thus, the attack at the BNP... 

Although inversion was not observed with the E. colt alkaline phosphatase, it has been observed for ribonucleases and many other hydrolytic enzymes and for most kinases transferring phospho groups from ATP. The difference lies in the existence of a phospho-enzyme intermediate in the action of alkaline phosphatase (see Eq. 12-38). Each of the two phosphotransferase steps in the phosphatase action apparently occurs with inversion. The simplest interpretation of all the experimental results is that phosphotransferases usually act by in-line -like mechanisms which may involve metaphosphate-ion-like transition states that are constrained to react with an incoming nucleophile to give inversion. An adjacent attack with pseudorotation would probably retain the original configuration and is therefore excluded. 

The most important chemical function of Zn2+ in enzymes is probably that of a Lewis acid providing a concentrated center of positive charge at a nucleophilic site on the substrate/ This role for Zn2+ is discussed for carboxypeptidases (Fig.12-16) and thermolysin, alkaline phosphatase (Fig. 12-23),h RNA polymerases, DNA polymerases, carbonic anhydrase (Fig. 13-1),1 class II aldolases (Fig. 13-7), some alcohol dehydrogenases (Fig. 15-5), and superoxide dismutases (Fig.16-22). Zinc ions in enzymes can often be replaced by Mn2+, Co2+, and other ions with substantial retention of catalytic activity/ ... 

In enzymes, the most common nucleophilic groups that are functional in catalysis are the serine hydroxyl—which occurs in the serine proteases, cholinesterases, esterases, lipases, and alkaline phosphatases—and the cysteine thiol—which occurs in the thiol proteases (papain, ficin, and bromelain), in glyceraldehyde 3-phosphate dehydrogenase, etc. The imidazole of histidine usually functions as an acid-base catalyst and enhances the nucleophilicity of hydroxyl and thiol groups, but it sometimes acts as a nucleophile with the phos-phoryl group in phosphate transfer (Table 2.5). 

The serine group which becomes phosphorylated does not appear to possess any marked nucleophilic reactivity, nor is there any evidence that a histidine group participates as a general acid-general base catalyst. Rate constants for the nonenzymic hydrolysis of alkyl and aryl phosphate monoanions at 25° are in the range HP7 to 10-9 sec-1 (167), while the comparable alkaline phosphatase-catalyzed values (in this case they refer to dianions) are in the range 102 to 103 sec-1. Thus one has to account for a rate enhancement factor of 109 to 1012. Moreover, the... 

The enzymes used to generate reactive quinone methides often undergo inactivation by addition of this electrophile to essential nucleophilic amino acid side chains of the protein catalyst. This is a type of suicide enzyme inhibition.80 This was observed for the acid phosphatase and ribonuclease catalysts used to generate 43.76 79 Alkaline phosphatase has been used to remove the phosphate protecting group from a derivative of an o-difluoromethyl phenyl phosphate that was covalently attached to a solid support. Breakdown of the immobilized 4-hydroxybenzyl difluoride gives an immobilized quinone methide that, in principle, will react irreversibly with proteins and lead to their attachment to the solid support.81... 

The active sites of these enzymes can have a nitrogen ligand, usually as histidine (acid phosphatases and some protein phosphatases), a nucleophilic serine residue (alkaline phosphatases), a cysteine residue in which the thiol group can form a covalent species with the phosphate ester (protein phosphatases), or an aspartate-linked phosphate (plasma membrane ion pumps). The inhibitory form of vanadium is usually anionic vanadate V(V), but cationic vanadyl V(IV) has also shown strong inhibition of some types of phosphorylase reactions. Above neutral pH, speciation of vanadyl ions produces anionic V(IV) species capable of inhibition of enzymes in the traditional transition-state analogue manner [5],... 

Artificial enzymes with metal ions can also hydrolyze phosphate esters (alkaline phosphatase is such a natural zinc enzyme). We examined the hydrolysis of p-nitro-phenyfdiphenylphosphate (29) by zinc complex 30, and also saw that in a micelle the related complex 31 was an even more effective catalyst [118]. Again the most likely mechanism is the bifunctional Zn-OH acting as both a Lewis acid and a hydroxide nucleophile, as in many zinc enzymes. By attaching the zinc complex 30 to one or two cyclodextrins, we saw even better catalysis with these full enzyme mimics [119]. A catalyst based on 25 - in which a bound La3+ cooperates with H202, not water - accelerates the cleavage of bis-p-nitrophenyl phosphate by over 108-fold relative to uncatalyzed hydrolysis [120]. This is an enormous acceleration. 

The third example is a phosphoryl transfer enzyme, alkaline phosphatase. The active site of alkaline phosphatase contains two Zn + ions, with a separation of 3.9 A. One zinc center is used to bind the phosphate monoester substrate, the other to activate Ser-102 for nucleophilic attack on the phosphate group of the substrate via an associative mechanism, as shown... 

The Escherichia coli alkaline phosphatase (li coli AP) is the most extensively studied phosphatase, and perhaps the most studied two-metal ion catalyst.68,91 95 The AP-catalyzed reaction proceeds via an intermediate in which a serine residue (Ser-102 in E. coli AP) is phosphorylated. Thus, the stereochemical outcome of the overall reaction is retention. The hydrolysis of the intermediate by water to produce inorganic phosphate competes with phosphoryl transfer to other acceptors such as alcohols or nucleophilic buffers if such are present in solution. The rate-limiting step... 

The AP from E. coli contains two Zn2+ ions and one Mg2+ ion in the active site.68,91 The Zn ions play the most direct roles in catalysis the Mg2+ has been suggested to function as the provider of the general base that deprotonates the Ser nucleophile, in the form of a Mg-coordinated hydroxide.98 All known alkaline phosphatases have this conserved three metal ion center, as well as an arginine residue (Arg-166 in E. coli AP) that plays a role in binding and probably in transition state stabilization (Fig. 18). 

Treatment of thymidine 5 -phosphorodiamidate (54) with pyrophosphate in anhydrous DMF leads to the formation of P -(thymidine-5 )-P -aminotriphosphate (55) in good yield. The compound is sufficiently stable to be purified on DEAE-Sephadex at low temperature. In acid, conversion into TTP is quantitative, but in ammonia, thymidine-5 -phosphoramidate and pyrophosphate are formed. Treatment with alkaline phosphatase cleaves (55) to the phosphoramidate. Nucleophilic attack takes place very easily in anhydrous DMF at ambient temperature, (55) is in equilibrium with (56) and pyrophosphate. 

The presence of a metal on a phosphoryl transferring-enzyme provides no assurance that the metal is directly involved in phosphoryl transfer. Thus with alkaline phosphatase, no direct interactions of Cl- with enzyme bound Zn2+ (69) or water with enzyme-bound Mn2+ (70) were detected by nuclear relaxation. Similarly no direct interaction of phosphate with enzyme bound Co2+ (71) or Mn2+ (71, 72) was detected by 31P nuclear relaxation. A Mn2+ to phosphate distance of 7.3 A was calculated from NMR data on the inactive Mn2+-enzyme (73) indicative of a second sphere complex. These results are in accord with crystallographic data on the enzyme which at 7.7 A resolution indicate that substrates cannot easily gain direct access to the metal site (74). More recent proton relaxation studies with the Cu2+ enzyme, which retains 5% of the activity, indicate the presence of a rapidly exchanging axial hydroxyl ligand on Cu2+ suggesting that the active metals may promote the nucleophilicity of the water molecule which is to attack the phosphorus (75). 

Insight into the role of the serine nucleophile in the catalytic cycle of alkaline phosphatase was gained through studies of the bis(4-nitrophenyl) phosphate reactivity of a mononuclear Zn(II) complex supported by the (S)-1 -(2-hydroxy-2-phe-nylethyl)- 1,4,7,10-tetrazacyclodecane ligand (Scheme 33).226 In aqueous solution, this complex exhibits a pKa value for the zinc-bound alkoxide moiety of 7.30 + 0.02. 

Inclusion of an alcohol appendage in the L24 ligand framework (Fig. 52) results in a reaction pathway for bis(4-nitrophenyl) phosphate hydrolysis that involves initial nucleophilic attack of a zinc-bound alkoxide moiety on the substrate to give a phosphorylated intermediate (Scheme 34).239 This intermediate, similar to the phosphorylated serine intermediate proposed in the catalytic cycle of alkaline phosphatase, is subsequently attacked by a Zn-OH moiety to yield 4-nitrophenyl... 

In some kinases, such as nucleoside diphosphate kinase, " an intermediate step is the phosphoryl transfer to a group belonging to the enzyme, as happens in ATPase and as was discussed in detail for alkaline phosphatase (Section V.B). In other kinases the phosphoryl transfer occurs directly from the donor to the acceptor in a ternary complex of the enzyme with the two substrates.Often metal ions like magnesium or manganese are needed. These ions interact with the terminal oxygen of the ATP molecule, thus facilitating the nucleophilic attack by the acceptor. The metal ion is often associated with the enzyme. For mechanistic schemes, see the proposed mechanism of action of alkaline phosphatase, especially when a phosphoryl enzyme intermediate is involved. 

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