Electrostatic effect upon association of reduced nicotinamide adenine dinucleotide and equine liver alcohol dehydrogenase

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aliesterase activity in normal and postheparin human blood sera.

An enzyme with characteristics typical of aliesterase has been found in human blood serum using a gas solid chromatographic assay technique. This conflicts with the findings of several authors that aliesterase is absent in the human blood. Another aliesterase is released into the blood stream after intravenous administration of heparin. Partial purification of the aliesterase in normal (preheparin) and postheparin sera was effected by column chromatography using CM- and DEAE-Sephadex. The preheparin aliesterase and postheparin aliesterase have different pH optima of 7.0 and 8.5 respectively. The preheparin aliesterase activity was very sensitive to sodium fluoride and insensitive to a negatively charged detergent, sodium lauryl sulfate, unlike the postheparin esterase which was highly sensitive to sodium lauryl sulfate and comparatively less sensitive to sodium fluoride.     

An essential ionized acid group in sodium channels.

Several recent experiments demonstrate the presence of an essential negatively charged acid group within sodium channels. Sodium permeability titrates away at low pH as if controlled by an acid with a voltage-dependent apparent pKa in the range between 5 and 6. The alkali ion permeability sequence of the channel is best explained by interactions between the cations and a strong negative charge in the channel. Block of sodium currents by a variety of metal and organic cations again points to a cation-coordinating site in the channel. The "blocking cations" and protons also oppose the bindings of tetrodotoxin and saxitoxin. The negative charge in the channel seems to be essential in selecting appropriate cations and in lowering their activation energy for permeation. The same charge seems to form part of the toxin receptor. At present this charged group is the chemical group known to be associated with sodium channels.--Hille B. An essential ionized acid group in sodium channels.     

Interaction of polycations with cell-surface negative charges of epithelial cells.

The interaction between various polycations and cultured glomerular epithelial cells was studied by cell electrophoresis. It was shown that the glomerular epithelial cell presents a negatively charged surface which imparts a zeta potential of -29.0 +/- 1.5 mV at the peripheral layer of the plasma membrane. The pH at which the GEC charge became 50% reduced (pKa) was determined to be 3.0. A variety of polycations of various sizes and fixed and flexible geometries were tested for their capacity to neutralize the cell charge. All the polycations except cytochrome c and lysozyme were capable of completely neutralizing the cell. Cytochrome c could maximally neutralize only 50% of charge and lysozyme only 72% of charge. However, reduced and 'relaxed' molecules of cytochrome c and lysozyme efficiently neutralized the cell surface, as did larger sized 'flexible' polylysines. On the basis of these findings, we conclude that all polycations are not equal in their capacity to neutralize the cell surface. Flexible molecules in contrast to molecules with rigid structures were more effective in neutralizing the cell. This may likely be due to the exposure and availability of more cationic groups in a flexible molecule which results in stabilization of interaction with cells.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.

Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.     

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.     

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.     

Effects of electric charge on hydraulic conductivity of pulmonary interstitium

The hydraulic conductivity of pulmonary interstitium was measured in a short isolated segment of interstitium surrounding a large pulmonary artery (1-3 mm diam) of the rabbit. The flow rate of the following solutions was measured sequentially: normal saline, polycation protamine sulfate (0.08 mg/ml), cationic dextran (0.1 or 1.5%) or anionic dextran (0.1 or 1.5%), and hyaluronidase (testes, 0.02%) solution. The pH of all solutions was adjusted to 7.35-7.40. The ratios of the flow of protamine sulfate and cationic dextran to that of saline averaged 2.3 +/- 0.92 (SD, n = 7) and 3.0 +/- 1.2 (n = 6), respectively. The anionic dextran-to-saline flow ratio averaged 0.72 +/- 0.28 (n = 13). Flow increased in the presence of positively charged molecules and decreased in the presence of negatively charged molecules. At a lower pH of 5.0-6.0, only 0.1% cationic dextran had an effect on interstitial conductivity. Thus pulmonary interstitium at physiological pH has the properties of a negatively charged membrane. The increased interstitial conductivity caused by the positively charged molecules was not observed after treatment with hyaluronidase. These effects of electric charge on interstitial conductivity were partly attributed to the presence in the interstitium of negatively charged hyaluronan.     

Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria.

The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pKa of the uncoupler molecule. The absorption and fluorescene excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca-2+ stongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes. With mitochondrial membranes an effect of Ca-2+ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochrondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it. From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholiped moiety of the membrane, with its anionic group pointing to the aqueous solution.     

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.     

pKa of the mRNA cap-specific 2'-O-methyltransferase catalytic lysine by HSQC NMR detection of a two-carbon probe

We have characterized the side chain pKa for a single lysine analogue within a 316-residue protein containing 21 lysines and 1678 carbon atoms at natural isotope abundance. To do this, the single reactive cysteine of a K175C mutant of VP39 (the mRNA cap-specific 2'-O-methyltransferase from vaccinia virus) was modified to S-(beta-aminoethyl)cysteine (gamma-thialysine) using freshly prepared (13C)aziridine at room temperature. Modification was accompanied by the rescue of catalytic function at high specific activity. After the fastidious removal of the noncovalently protein-bound aziridine self-polymer using a novel chelating dialysis procedure, signals were monitored by HSQC NMR. Appropriately pH-shifting HSQC NMR peaks were identified in the (13C)aziridine-modified enzyme, corresponding to detection of the two covalently attached (13C)thioethylamino atoms. The identification was strengthened by comparison with the positions and pH shifts of spectral peaks for tripeptide controls, a small molecule aziridine self-polymer mimetic, and a cysteine-minus control enzyme. pH titration of the modified protein indicated an apparent pKa of 8.5, consistent with a perturbed pKa for the catalytic lysine and a model in which the surrounding charged groups direct the lysine epsilon-amino pKa via both local electrostatic environment and orbital directionality.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.     

Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles.

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.     

CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore

We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.     

Zinc and cadmium 5-aminolevulinate dehydratase. Metal-dependent pH profiles

Native 5-aminolevulinic acid dehydratase contains zinc ions, which are essential for the enzymatic activity. Replacement of zinc by cadmium yielded an active enzyme whose kinetic parameters (kkat and Km) are similar to those of the zinc enzyme in the neutral pH range. However, the pH profiles of kcat and Km were different due to different pKa values. Two groups both with pKa values of 6.5 in the free zinc enzyme, but with pKa values of 7.0 in the cadmium enzyme were calculated from plots of log (kcat/Km) versus pH. On the other hand, the enzyme-substrate complex is controlled by one acidic group (zinc pKa = 6.0, cadmium pKa = 6.4) and one basis group (zinc pKa = 8.2, cadmium pKa = 7.7) as calculated from plots of log kcat versus pH. The Arrhenius plots for kcat of the two enzymes show no significant difference, the free energies of activation are 77.1 kJ/mol for the zinc and 76.8 kJ/mol for the cadmium enzyme. From this and from previous work it is concluded that the metal ions are located near the active site and influence the ionisations of essential amino acid residues. From the pH profiles of the modifying reaction and inhibition by diethylpyrocarbonate a histidinyl residue is inferred as one of the ionisable groups of the active site.     

Regulation by Mg2+ and Ca2+ of mitochondrial membrane integrity: study of the effects of a cytosolic molecule and Ca2+ antagonists.

The coupling of aliphatic amines to agarose by the cyanogen bromide reaction yields isourea linkages which are positively charged at pH 7. The presence of these cationic sites in affinity gels causes significant non-specific adsorption of proteins. Serum albumin was found to bind to a number of derivatized gels which possessed these charged groups. The use of adipic dihydrazide as the leash moiety yielded affinity gels which were noncharged at pH 7. Serum albumin failed to adsorb to these gels. Beta-galactosidase from Escherichia coli was found to be sensitive to both ionic and hydrophobic groups in an affinity gel. A sample of active-site inhibited enzyme was found to bind to an affinity gel which contained both the cationic isourea and a phenyl structure in the leash. Thus it was concluded that the affinity purification of this enzyme has yet to be demonstrated. These studies dictate against the use of salt and pH gradients to desorb enzymes from affinity sorbents.     

Reactivities of neutral and cationic forms of 2,2''-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe.

The second-order rate constants (k) for the reactions of 2,2'-dipyridyl disulphide (pKa2,45) with 2-mercaptoethanol (pKa9.6) and with benzimidazol-2-ylmethanethiol (pKa values 5.6 and 8.3) were determined at 25 degrees C at I 0.1 by stopped-flow spectral analysis over a wide range of pH. These were used to calculate the pH-independent second-order rate constants (k) for the reactions of neutral 2,2'-dipyridyl disulphide and of its monocation with the 2-mercaptoethanol thiolate anion (associated pKa9.6) and with the benzimidazol-2-ylmethanethiol zwitterion (associated pKa5.6). For both thiolate ions, the rate-enhancement factor (kmonocation/kneutral disulphide) is about 1.5x10(3). The dependence on pH in acidic media of k for the reaction of 2,2'-dipyridyl disulphide with actinidin, the thiol proteinase from Actinidia chinensis, was shown to differ from the forms of pH-dependence observed for the analogous reactions with papain (EC 3.4.22.2) and ficin (3.4.22.3). The reactivity of the 2,2'-dipyridyl disulphide dication and its apparent sensitivity to the presence and location of a positive charge in the attacking thiol are discussed.     

Intracellular X-prolyl dipeptidyl peptidase from Lactococcus lactis spp. lactis: purification and properties

An X-prolyl dipeptidyl peptidase (EC 3.4.14.5) has been purified from a crude intracellular extract from Lactococcus lactis spp. lactis NCDO 763 by ion-exchange chromatography and gel filtration. One protein band was detected after electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulphate. The enzyme is a 190 kDa dimer composed of identical subunits. Optimal activity occurs at pH 8.5 and 40–45°C and the enzyme is inhibited by reagents specific for serine proteases, such as diisopropylfluorophosphate. The enzyme hydrolyzes p -nitroanilide- or β-naphthylamide-substituted X-Pro dipeptides, as well as β-casomorphin.     

Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.