Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom.

The hydrolysis reaction of ATP alpha S by snake venom phosphodiesterase is highly specific for the B diastereomer and proceeds with 88% retention of configuration at phosphorus. Since this enzyme also catalyzes the hydrolysis of the S enantimoer of O-p-nitrophenyl phenylphosphonothioate, the absolute configuration at A alpha of ATP alpha S (B) is assigned as the R configuration provided the two substrates are processed identically. A mechanism for the hydrolysis reactions catalzyed by the venom phosphodiesterase involving at least a single covalent phosphoryl-enzyme intermediate is in accord with this result.     

Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase.

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.     

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.     

Stereochemistry of hydrolysis by snake venom phosphodiesterase.

[18O]Adenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled [18O]adenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to [18O]adenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging [1,2-mu-18O] or nonbridging [1-18O] was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.     

The stereochemical course of hydrolysis catalysed by snake venom 5''-nucleotide phosphodiesterase.

Adenosine 5'-(S)-[16O,17O,18O]phosphate was pyrophosphorylated by the combined action of adenylate kinase and pyruvate kinase. The isotopomers of adenosine 5'-[alpha-16O,17O,18O]triphosphate were hydrolysed by venom 5'-nucleotide phosphodiesterase (Crotalus adamanteus) in H2(17)O. Analysis by 31P nuclear magnetic resonance spectroscopy of the resulting adenosine 5'-[16O,17O,18O]phosphate, after cyclization and esterification, showed that the hydrolysis occurs with retention of configuration at phosphorus. The most likely explanation of this observation is that the enzymic hydrolysis involves a double displacement at phosphorus with a covalent nucleotidyl--enzyme intermediate on the reaction pathway.     

Reaction of enzyme-bound 5-deazaflavin with peroxides. Pyrimidine ring contraction via an epoxide intermediate

Reaction of peroxides with 5-deazaflavin bound to glucose oxidase, lactate oxidase, or D-amino acid oxidase results in the formation of 5-deazaflavin 4a, 5-epoxide. The reaction of D-amino acid oxidase with m-chloroperoxybenzoate is an exception since the reagent reacts rapidly with the protein moiety to form m-chlorobenzoate which then binds noncovalently near the unmodified coenzyme. Epoxide bound to glucose oxidase is converted to deazaFAD X X in a reaction similar to that observed previously with oxynitrilase and glycolate oxidase. With lactate oxidase the epoxide is quite stable in the absence of light. With D-amino acid oxidase, denaturation of the protein is accompanied by the release of the epoxide into solution where it decomposes in a manner similar to that observed with model epoxide compounds at neutral pH. Reaction of deazaFAD X X with phosphodiesterase and alkaline phosphatase yields deazariboflavin X X. The same compound has been formed in model studies by exposing 5-deazariboflavin 4a,5-epoxide to alkaline conditions. Structural studies indicate that this reaction involves contraction of the pyrimidine ring to yield 4-ribityl-6,7-dimethyloxazolo[ 4,5-b ]quinolin-2(4H)-one. Model reaction studies are consistent with a mechanism initiated by alkaline hydrolysis of the pyrimidine ring at position 4 followed by two additional steps which proceed at neutral pH. A similar mechanism for the enzyme reactions appears likely since analogous intermediates are detected in the glycolate oxidase and the model reactions. The results suggest that position 4 of the coenzyme in oxynitrilase, glycolate oxidase, and glucose oxidase must be accessible to solvent and that the protein moiety must facilitate the initial hydrolysis of the pyrimidine ring since the enzyme reactions occur at neutral pH. Failure to observe formation of deazaFMN X X with lactate oxidase is attributed, at least in part, to the inaccessibility of the pyrimidine ring to solvent.     

A simple method for preparation of snake venom phosphodiesterase almost free from 5'-nucleotidase.

A simple method, involving NAD+-Sepharose chromatography, was developed for the preparation of snake venom phosphodiesterase (EC 3.1.4.1) almost free from 5'-Nucleotidase (EC 3.1.3.5). Using an NAD+-Sepharose 4B column, phosphodiesterase was eluted in the unadsorbed fraction, whereas 5'nucleotidase was strongly adsorbed. The latter enzyme was desorbed when 0.2 M sodium bicarbonate buffer containing 1mM beta-NADH was used as a solvent. The affinity column could be used at least four times without any decrease of potency, and the method was applicable for the preparation of phosphodiesterase from the venoms of rattlesnake (Crotalus adamanteus) and Japanese mamushi (Agkistrodan halys blomhoffi).     

Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom

A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of -mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 59-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5–9.5 and the optimum temperature was 60°C, with activity being rapidly lost within 1 min at 70°C. The K_m of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and -mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.     

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.     

Platelet cyclic 3':5'-nucleotide phosphodiesterase released by thrombin and calcium ionophore.

Contact of rat platelets with thrombin or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in the secretion of adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterases. Significant association of calcium with platelets occurred during platelet surface contact with thrombin. Thrombin concentration to induce association of calcium virtually agreed with that to release the enzyme. The finding that A-23187 (5 to 20 muM) also provoked a rapid and marked association of extracellular calcium with platelets suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between platelet and calcium. Two different phosphodiesterases, a relatively specific cyclic AMP and a relatively specific cyclic GMP phosphodiesterase were secreted from platelets into the plasma in soluble form. The amounts of the phosphodiesterases secreted were dose- or time-dependent on thrombin (0.1 to 2 units) or A-23187 (5 to 20 muM) within 30 min. The enzyme release by thrombin was completely inhibited by heparin but the release by A-23187 was not. The two phosphodiesterases secreted seemed to correspond to the two enzymes isolated from platelet homogenates in many respects. Rat platelets contained, at least, three cyclic 3':5'-nucleotide phosphodiesterases, namely, two relatively specific cyclic AMP phoshodiesterases and a relatively specific cyclic GMP phosphodiesterase which were clearly separated from each other by Sepharose 6B or DEAE-cellulose column chromatography or sucrose gradient centrifugation. The two platelet cyclic AMP phosphodiesterase (Mr = 180,000 and 280,000) had similar apparent Km values of 0.69 and 0.75 muM with different sedimentation coefficient values of 4.9 S and 7.1 S, respectively. They did not hydrolyze cyclic GMP significantly. A cyclic GMP phosphodiesterase (Mr - 260,000) exhibited abnormal kinetics for cyclic GMP with an apparent Km value of 1.5 muM and normal kinetics for cyclic AMP with a Km of 300 muM. The properties of a platelet cyclic AMP phosphodiesterase (Mr = 180,000) and a platelet cyclic GMP phosphodiesterase were found to agree with those of the two phosphodiesterases released from platelets by thrombin or A-23187. Depletion of extracellular calcium by an addition of citrate, EDTA, or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the blood or platelet suspension resulted in a loss of the activity of the smaller form of platelet cyclic AMP phosphodiesterase (Mr = 180,000) and addition of calcium restored the activity of this cyclic AMP phosphodiesterase. Thus, calcium seemed to be involved in the mechanism of an occurrence of this smaller form of cyclic AMP phosphodiesterase as well as the secretion of this enzyme. Contact of human platelets with thrombin also resulted in the secretion of cyclic nucleotide phosphodiesterase which was dependent on the concentration of calcium. No species difference was observed in this respect.     

Methanol esterification reactions catalyzed by snake venom and bovine intestinal 5'-nucleotide phosphodiesterases. Formation of nucleoside 5'-monophosphate methyl esters from guanosine 5'-triphosphate and other nucleoside 5'-polyphosphates.

It is not known whether the enzymes 5'-nucleotide phosphodiesterase/nucleotide pyrophosphatase (EC 3.1.4.1/EC 3.6.1.9) catalyze the transfer of nucleotides to acceptors other than water. We have investigated the action of snake venom and bovine intestinal mucosa phosphodiesterases on nucleoside 5'-polyphosphates in the presence of methanol. In those conditions, GTP was converted by snake venom phosphodiesterase to a mixture of GMP and another compound with a different retention time in reverse-phase high-performance liquid chromatography. That compound, by ultraviolet, 1H- and 13C-nuclear magnetic resonance spectroscopic analysis, and by enzyme analysis, was characterized as the methyl ester of GMP (GMP-OMe). The molar fraction [GMP-OMe]/[GMP + GMP-OMe] formed was higher than the molar fraction of methanol as a solvent in reaction mixtures. Similar reactions took place at comparable rates with snake venom and bovine intestinal mucosa phosphodiesterases using several nucleoside 5'-polyphosphates as substrates. The ability of 5'-nucleotide phosphodiesterases to catalyze transfer reactions to a non-water acceptor is relevant to the mechanism of the enzymes, to their use as analytical tools, and to their possible use/role in the preparative/in vivo synthesis of nucleotide esters.     

Particulate cyclic 3',5'-nucleotide phosphodiesterase and calmodulin of cardiac muscle

Cyclic AMP and cyclic GMP phosphodiesterase and calmodulin were measured in purified subcellular fractions of cardiac muscle. Phosphodiesterase activity solubilized by sonication of the nuclear fraction yielded a major 6.6 S form which was calcium-sensitive and cyclic GMP-specific. Phosphodiesterase activity occurring in the nuclear fraction could be further enriched by subfractionation on sucrose density gradients in the presence of MgCl2.     

Fluorometric assay of serum 5'-nucleotide phosphodiesterase in normal humans and cancer patients

A sensitive method of assaying serum 5′-nucleotide phosphodiesterase (PDase) is described. A preliminary study did not find significant differences between the levels of PDase in cancer patients and those of normal human sera.