Assay of covalent intermediate of 5'-nucleotide phosphodiesterase

We report a new procedure for isolating a covalent phosphoryl enzyme (diester) intermediate of bovine intestinal 5'-nucleotide phosphodiesterase. The convenience of the procedure makes it possible to determine effects of reaction conditions on the yield of covalent intermediate. Under optimum conditions, using [methyl-3H]deoxythymidine 5'-triphosphate as substrate, more than 50% of the enzyme is recovered as thymidylyl enzyme, a 10-fold increase in yield over the previous procedure (M. Landt and L. G. Butler, 1978, Biochemistry 17, 4130-4135). Yields of thymidylyl enzyme were maximal at pH 4, whereas optimum catalytic activity is observed at pH greater than 9.     

A family of phosphohydrolases from bovine intestinal mucosa: 5'-nucleotidase

Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase.     

Amino acid sequence of the active site peptide of bovine intestinal 5'-nucleotide phosphodiesterase and identification of the active site residue as threonine

We report here the identification of the amino acid residue which forms the covalent intermediate in the catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase and the sequence of the neighboring amino acids. The active site of 5'-nucleotide phosphodiesterase was labeled using thymidine 5'-[alpha-32P]triphosphate as substrate. A single labeled cyanogen bromide peptide was isolated using reversed-phase high performance liquid chromatography. After subdigestion with endoproteinase Lys-C and chymotrypsin, the entire amino acid sequence of the 60-residue active site peptide was obtained using automated Edman degradation. All of the radioactivity of the active site peptide was localized to a hexapeptide with sequence Thr-Phe-Pro-Asn-His-Tyr. Phosphoamino acid analysis of this peptide indicated that the labeled residue was threonine. We are not aware of any other enzymes in which threonine is phosphorylated as a covalent intermediate in the catalytic mechanism.     

Enzymic hydrolysis of phosphonate esters. Reaction mechanism of intestinal 5'-nucleotide phosphodiesterase.

The mechanism of bovine intestinal 5'-nucleotide phosphodiesterase was investigated by determining kinetic constants of systematically varied substrates, with emphasis on esters of phosphonic acids (which have much higer Vmax values than conventional phosphodiester substrates), and by pre-steady-state kinetics using bis(4-nitrophenyl) phosphate as substrate. The results suggest a ping-pong type mechanism, with participation of a covalent enzyme intermediate.     

Alkaline phosphatase and 5'-nucleotide phosphodiesterase from bovine intestine are cross-reactive

Polyclonal antibodies to native alkaline phosphatase and to native 5'-nucleotide phosphodiesterase were found to strongly cross-react with both enzymes. The antibodies also cross-react with both denatured enzymes, with glycopeptides from 5'-nucleotide phosphodiesterase, and with the oligosaccharides remaining after Pronase E digestion of the phosphodiesterase. They do not cross-react with either enzyme after their oligosaccharides have been modified or removed by periodate or trifluoromethanesulfonic acid treatment. Antibodies to denatured 5'-nucleotide phosphodiesterase do not bind to the native phosphodiesterase or alkaline phosphatase but do cross-react with denatured alkaline phosphatase even after removal or modification of the carbohydrate moieties. These results suggest that antibodies to denatured 5'-nucleotide phosphodiesterase may recognize amino acid sequence homology between alkaline phosphatase and 5'-nucleotide phosphodiesterase. However, antibodies to native enzymes apparently recognize cross-reactive determinants of the native enzymes which are carbohydrate in nature. This is the first report of antimammalian alkaline phosphatase antibodies which recognize the carbohydrate moieties of the enzyme.     

A nucleotide phosphodiesterase with preference for 2',5'-phosphodiester bonds from Ehrlich ascites carcinoma

We have previously reported that many tumor cell lines express a 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) with properties clearly distinguishable from enzymes of normal tissues (Biochim. Biophys. Acta (1988) 966, 99-106). Such an enzyme with 5'-nucleotide phosphodiesterase activity was purified from Ehrlich ascites carcinoma by measuring the cleavage of thymidine 5'-monophosphate p-nitrophenyl ester (TMP-NP). The enzyme is a soluble protein, has a pH optimum of 7.5, and the molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 67 kDa. The enzyme does not hydrolyze other chromogenic substrates for phosphodiesterases, nor pyrophosphate bond of various nucleotides which are cleaved by 5'-nucleotide phosphodiesterases of normal tissues. But, it hydrolyzes dinucleotides to form 5'-phosphates, and is more active on 2',5'- than on 3',5'-phosphodiester bonds. These results indicate that the TMP-NP splitting enzyme in Ehrlich ascites carcinoma cells is a 2',5'-phosphodiesterase.     

Phosphorylated threonine as the covalent intermediate in snake venom phosphodiesterase

The covalent intermediate of snake venom phosphodiesterase has been isolated using thymidine 5'-[alpha-32P]triphosphate as substrate. Phosphoamino acid analysis of the labeled enzyme demonstrates that threonine is the active site residue forming the covalent intermediate. 5'-Nucleotide phosphodiesterase is the first enzyme reported to have an active site threonine forming a covalent intermediate.     

Energetics of alpha-chymotrypsin-mediated hydrolysis of a strained cyclic ester

Hydrolysis of omicron-hydroxy-alpha-toluenesulfonic acid sultone (sultone II) is mediated by alpha-chymotrypsin. Sultone II is a highly strained cyclic ester substrate that forms a covalent intermediate with the enzyme and is therefore expected to release ring-strain energy upon formation of the sulfonyl enzyme species. It is found that the equilibrium constant for forming the covalent intermediate from the Michaelis complex is quite modest (K2 = 16.4), suggesting that perhaps the strain energy is not released in the ring-cleavage event. The implied retention of chemical (strain) energy by the sulfonyl enzyme species raises the question of the means by which the enzyme avoids expression of strain energy and the implications of this effect in the catalytic sequence. High-pressure liquid chromatography (HPLC) rate data demonstrate facile reversion of sulfonyl enzyme to the Michaelis complex, and that reversion is preferred over hydrolysis of the covalent intermediate. pH-independent rate and equilibrium constants are derived for the alpha-chymotrypsin-mediated hydrolysis of sultone II, and pKa values for groups on the enzyme are reported that are consistent with literature values obtained from analysis of nonspecific substrate hydrolysis by the enzyme.     

Binding, tuning and mechanical function of the 4-hydroxy-cinnamic acid chromophore in photoactive yellow protein.

The bacterial photoreceptor protein photoactive yellow protein (PYP) covalently binds the chromophore 4-hydroxy coumaric acid, tuning (spectral) characteristics of this cofactor. Here, we study this binding and tuning using a combination of pointmutations and chromophore analogs. In all photosensor proteins studied to date the covalent linkage of the chromophore to the apoprotein is dispensable for light-induced catalytic activation. We analyzed the functional importance of the covalent linkage using an isosteric chromophore-protein variant in which the cysteine is replaced by a glycine residue and the chromophore by thiomethyl-p-coumaric acid (TMpCA). The model compound TMpCA is shown to weakly complex with the C69G protein. This non-covalent binding results in considerable tuning of both the pKa and the color of the chromophore. The photoactivity of this system, however, was strongly impaired, making PYP the first known photosensor protein in which the covalent linkage of the chromophore is of paramount importance for the functional activity of the protein in vitro. We also studied the influence of chromophore analogs on the color and photocycle of PYP, not only in WT, but especially in the E46Q mutant, to test if effects from both chromophore and protein modifications are additive. When the E46Q protein binds the sinapinic acid chromophore, the color of the protein is effectively changed from yellow to orange. The altered charge distribution in this protein also results in a changed pKa value for chromophore protonation, and a strongly impaired photocycle. Both findings extend our knowledge of the photochemistry of PYP for signal generation.     

The electrostatic driving force for nucleophilic catalysis in L-arginine deiminase: a combined experimental and theoretical study

L-arginine deiminase (ADI) catalyzes the hydrolysis of L-arginine to form L-citrulline and ammonia via two partial reactions. A working model of the ADI catalytic mechanism assumes nucleophilic catalysis by a stringently conserved active site Cys and general acid-general base catalysis by a stringently conserved active site His. Accordingly, in the first partial reaction, the Cys attacks the substrate guanidino C zeta atom to form a tetrahedral covalent adduct, which is protonated by the His at the departing ammonia group to facilitate the formation of the Cys- S-alkylthiouronium intermediate. In the second partial reaction, the His activates a water molecule for nucleophilic addition at the thiouronium C zeta atom to form the second tetrahedral intermediate, which eliminates the Cys in formation of the L-citrulline product. The absence of a basic residue near the Cys thiol suggested that the electrostatic environment of the Cys thiol, in the enzyme-substrate complex, stabilizes the Cys thiolate anion. The studies described in this paper explore the mechanism of stabilization of the Cys thiolate. First, the log(k(cat)/K(m)) and log k(cat) pH rate profiles were measured for several structurally divergent ADIs to establish the pH range for ADI catalysis. All ADIs were optimally active at pH 5, which suggested that the Cys pKa is strongly perturbed by the prevailing electrostatics of the ADI active site. The p K a of the Bacillus cereus ADI (BcADI) was determined by UV-pH titration to be 9.6. In contrast, the pKa determined by iodoacetamide Cys alkylation is 6.9. These results suggest that the negative electrostatic field from the two opposing Asp carboxylates perturbs the Cys pKa upward in the apoenzyme and that the binding of the iodoacetamide (a truncated analogue of the citrulline product) between the Cys thiol and the two Asp carboxylates shields the Cys thiol, thereby reducing its pKa. It is hypothesized that the bound positively charged guanidinium group of the L-arginine substrate further stabilizes the Cys thiolate. The so-called "substrate-assisted" Cys ionization, first reported by Fast and co-workers to operate in the related enzyme dimethylarginine dimethylaminohydrolase [Stone, E. M., Costello, A. L., Tierney, D. L., and Fast, W. (2006) Biochemistry 45, 5618-5630], was further explored computationally in ADI by using an ab initio quantum mechanics/molecular mechanics method. The energy profiles for formation of the tetrahedral intermediate in the first partial reaction were calculated for three different reaction scenarios. From these results, we conclude that catalytic turnover commences from the active configuration of the ADI(L-arginine) complex which consists of the Cys thiolate (nucleophile) and His imidazolium ion (general acid) and that the energy barriers for the nucleophilic addition of Cys thiolate to the thiouronium C zeta atom and His imidazolium ion-assisted elimination from the tetrahedral intermediate are small.     

On the mechanism of action of bovine intestinal mucosa 5'-nucleotide phosphodiesterase. Stereochemical evidence for a nucleotidyl-enzyme intermediate

The diastereoisomers of adenosine 5'-O-phosphorothioate O-methyl ester have been synthesised. Only the Sp diastereoisomer is a substrate for the 5'-nucleotide phosphodiesterase from bovine intestinal mucosa. The previously unidentified enantiomer of 4-nitrophenyl phenyl phosphonothioate hydrolysed by the enzyme is shown to have the Sp configuration. Digestion of the Sp diastereoisomer of adenosine 5'-O-phosphorothioate O-methyl ester by the enzyme in 18O-labelled water gave 18O-labelled adenosine 5'-O-phosphorothioate which was stereochemically analysed by methylation and subsequent 31P-NMR spectroscopy and shown to possess the Sp configuration. Thus the enzyme-catalysed cleavage proceeded with retention of configuration at phosphorus, presumably via a double-displacement mechanism. This provides strong evidence for the existence of a nucleotidyl-enzyme intermediate on the reaction pathway.     

Specificity and pH dependence for acylproline cleavage by prolidase

Catalytic pH dependence for the hydrolytic activity of the enzyme prolidase with a series of dipeptide substrates is found to be generally bell-shaped (kcat/Km) or simple sigmoidal (kcat). An enzymic residue with a pKa value of 6.6 is found to be critically involved in the catalytic mechanism, as is the substrate amino group. Significant catalysis at a pH of 6.6 is also observed for prolidase with (alkylthio)acetylprolines and with haloacetylprolines. A reverse-protonation state mechanism for substrate binding and activation is postulated, involving a chelative interaction of the aminoacylamide portion of substrate with a strongly Lewis-acidic active site metal ion.     

Effect of pH on substrate and inhibitor kinetic constants of human liver alanine aminopeptidase. Evidence for two ionizable active center groups.

The presence of at least two ionizable active center groups has been detected by a study of the effect of pH upon catalysis of hydrolysis of L-alanyl-beta-naphthylamide by human liver alanine aminopeptidase and upon the inhibition of hydrolysis by inhibitors and substrate analogs. Octanoic acid, octylamine, and peptide inhibitors have been found to be competitive inhibitors and are therefore thought to bind the active center. L-Phe was previously shown to bind the active center since it was found to be a competitive inhibitor of the hydrolysis of tripeptide substrates (Garner, C. W., and Behal, F. J. (1975), Biochemistry 14, 3208). A plot of pKm vs. pH for the substrate L-Ala-beta-naphthylamide showed that binding decreased below pH 5.9 and above 7.5, the points at which the theoretical curve undergoes an integral change in slope. These points are interpreted as the pKa either of substrate ionizable groups or binding-dependent enzyme active center groups. Similar plots of pKm vs. pH for L-alanyl-p-nitroanilide (as substrate) and pKi vs. pH for L-Leu-L-Leu-L-Leu and D-Leu-L-Tyr (as inhibitors) gave pairs fo pKa values of 5.8 and 7.4, 6.0 and 7.5, and 5.7 and 7.5, respectively. All the above substrates (and D-Leu-L-Tyr) have pKa values near 7.5; therefore, the binding-dependent group with a pKa value near 7.5 is possibly this substrate group. Similar plots of pKi vs. pH for the inhibitors L-Phe, L-Met, L-Leu, octylamine, and octanoic acid had only one bending point at 7.7, 7.6, 7.4, 6.3, and 5.9, respectively. Amino acid inhibitors, octylamine, and octanoic acid have no groups with pKa values between 5 and 9. These data indicate that there are two active center ionizable groups with pKa values of approximately 6.0 and 7.5 which are involved in substrate binding or inhibitory amino acid binding but not in catalysis since Vmax was constant at all pH values tested.     

Leaving group dependence and proton inventory studies of the phosphorylation of a cytoplasmic phosphotyrosyl protein phosphatase from bovine heart.

The kcat and Km values for the bovine heart low molecular weight phosphotyrosyl protein phosphatase catalyzed hydrolysis of 16 aryl phosphate monoesters and of five alkyl phosphate monoesters having the structure Ar(CH2)nOPO3H2 (n = 1-5) were measured at pH 5.0 and 37 degrees C. With the exception of alpha-naphthyl phosphate and 2-chlorophenyl phosphate, which are subject to steric effects, the values of kcat are effectively constant for the aryl phosphate monoesters. This is consistent with the catalysis being nucleophilic in nature, with the existence of a common covalent phosphoenzyme intermediate, and with the breakdown of this intermediate being rate-limiting. In contrast, kcat for the alkyl phosphate monoesters is much smaller and the rate-limiting step for these substrates is interpreted to be the phosphorylation of the enzyme. A single linear correlation is observed for a plot of log (kcat/Km) vs leaving group pKa for both classes of substrates at pH 5.0: log (kcat/Km) = -0.28pKa + 6.88 (n = 19, r = 0.89), indicating a uniform catalytic mechanism for the phosphorylation event. The small change in effective charge (-0.28) on the departing oxygen of the substrate is similar to that observed in the specific acid catalyzed hydrolysis of monophosphate monoanions (-0.27) and is consistent with a strong electrophilic interaction of the enzyme with this oxygen atom in the transition state. The D2O solvent isotope effect and proton inventory experiments indicate that only one proton is "in flight" in the transition state of the phosphorylation process and that this proton transfer is responsible for the reduction of effective charge on the leaving oxygen.     

Repressible extracellular phosphodiesterases showing cyclic 2'',3''- and cyclic 3'',5''-nucleotide phosphodiesterase activities in Neurospora crassa.

Two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type Neurospora crassa and purified. The two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. This enzyme had the same electrophoretic mobility as repressible acid phosphatase. The enzyme designated repressible cyclic phosphodiesterase showed pH optima of 3.2 to 4.0 with a cyclic 3',5'-AMP substrate and 5.0 to 5.6 with a cyclic 2',3'-AMP substrate. Repressible cyclic phosphodiesterase was activated by MnCl2 and CoCl2 with cyclic 2',3'-AMP as substrate and was slightly activated by MnCl2 with cyclic 3',5'-AMP. The enzyme hydrolyzed cyclic 3',5'- and cyclic 2',3'-nucleotides, in addition to bis-rho-nitrophenyl phosphate, but not certain 5' -and 3'-nucleotides. 3'-GMP and 3'-CMP were hydrolyzed less efficiently. Mutant strains A1 (nuc-1) and B1 (nuc-2), which cannot utilize RNA or DNA as a sole source of phosphorus, were unable to produce repressible cyclic phosphodiesterase. The wild type (74A) and a heterocaryon between strains A1 and B1 produced the enzyme and showed growth on orthophosphate-free media containing cyclic 2',3'-AMP or cyclic 3',5'-AMP, whereas both mutants showed little or no growth on these media.     

Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.     

Substrate specificity and protonation state of Escherichia coli ornithine transcarbamoylase as determined by pH studies. Binding of carbamoyl phosphate

Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions. The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases. Two ionizing groups are involved in carbamoyl phosphate binding. Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM. Protonation of a second group further enhances binding by roughly another order of magnitude. This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex. However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis. The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2. Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp. Together with earlier kinetic results (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine.     

Effect of pH on the transient reduction of pig-plasma benzylamine oxidase by benzylamine derivatives.

1. The transient kinetics of reduction of the 470-nm absorption band in benzylamine oxidase by substrate at different pH values between 6 and 10 have been studied by stopped-flow techniques, and substituent effects on kinetic parameters for the reduction process have been examined using a series of ring-substituted benzylamine derivatives as the substrates. 2. Reduction of the enzyme by substrate takes place in two kinetically distinguishable steps, with the intermediate formation of an enzyme-substrate complex in which the substrate appears to be covalently bound through its amino group to the prosthetic group of the enzyme, possibly in the form of an amine-pyridoxal Schiff-base. 3. The apparent stability of the enzyme-substrate complex shows no obvious dependence on the electronic properties of the amine substrates, but is strongly pH-dependent in a way suggesting that substrate-binding involves the non-protonated amines, exclusively, and requires the presence of the acid form of an ionizing group in the enzyme with apparent pKa of 8.8. 4. Reduction of the enzymatic 470-nm chromophore and release of the aldehyde product of the catalytic process are rate-limited by the same monomolecular reaction step involving the enzyme-substrate complex. Rate constants for the rate-limiting reaction exhibit no significant dependence on pH between 6 and 10, but correlate with Hammett sigma-values for the ring-substituted benzylamine derivatives tested, yielding a phi-value of + 0.3.     

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.     

Mechanistic aspects of the low-molecular-weight phosphatase activity of the calmodulin-activated phosphatase, calcineurin

Product and substrate analogs have been employed as inhibitors of the low-molecular-weight phosphatase activity of calcineurin, a calmodulin-activated protein phosphatase. Product inhibition kinetics demonstrate that both products, para-nitrophenol and inorganic phosphate, inhibit para-nitrophenyl phosphate hydrolysis in a competitive manner. Inorganic phosphate is a linear competitive inhibitor, whereas the inhibition by para-nitrophenol is more complex. An analog of para-nitrophenol, pentafluorophenol, was found to be a linear competitive inhibitor. These patterns indicate a rapid equilibrium random kinetic mechanism for calcineurin. This mechanism suggests that calcineurin does not generate a phosphoryl enzyme during its catalytic reaction. Application of sulfate analogs indicates that binding of substrate occurs via the phosphoryl moiety. It is suggested that binding is a function of the affinity of ligand for the metal ion involved in calcineurin action. The dependence of the kinetic parameters of calcineurin upon pH was examined to provide information concerning the role of protonation in the activity and specificity of calcineurin. Log (VM) versus pH data for two low-molecular-weight substrates, para-nitrophenyl phosphate and tyrosine-O-phosphate, reveal a pKa value for the enzyme-substrate complex. Analysis of log (VM/KM) data yields a pKa value for the free enzyme of 8.0. Protonation of the phenolic leaving group during hydrolysis is not the rate-limiting step in calcineurin catalysis.