Purification and Kinetic Characterisation of Lactate Dehydrogenase from Dioscorea cayenensis Tuber

Lactate dehydrogenase from yellow yam tuber (Dioscorea cayenensis Lam.) was isolated and purified using various chromatographic methods and electrophoresis. Only one form of the enzyme obtained, which obeyed Michaelis-Menten kinetics, was activated by Mg²⁺ and Ca²⁺ and inhibited by nucleotides and PEP. AMP, which activated the enzyme in the direction of pyruvate reduction, inhibited it in the direction of lactate oxidation. The enzyme is specific for pyruvate L-lactate and uses only NADH and NAD⁺ as the electron carriers. Polyacrylamide gel electrophoresis showed single band of lactate dehydrogenase activity. The average molecular mass obtained for the enzyme was 160 ± 1.2 kDa, while SDS gel electrophoresis indicated a dimer for the enzyme protein. The enzyme is very stable when frozen but its activity was hardly detectable when the tubers were stored in a well aerated place.     

Delimitation of physiological regions in yam tubers (Dioscorea sp.) and distribution pattern of saccharide degrading enzymes,cell sap pH and protein in these regions

Physiological regions of yam tubers were morphologically defined in different specie into ‘Head’, ‘Middle’ and ‘Tail’, while the limits of these regions were studied using phosphorylase activity. Variation in enzyme activity, pH and protein concentration was found in different regions of the tubers. Old yam tubers had significantly higher activities of saccharide degrading enzymes, hexokinase, phosphorylase, glucose-6-phosphate dehydrogenase, phosphofructokinase and pyruvate kinase, than the new tubers. However, activity of phosphofructokinase in newD. rotundata was higher than that of old tuber. The high activity of phosphorylase in different regions of all the yam tubers examined indicates a very important role of this enzyme in starch degradation inDioscorea species. The measured pH and protein concentration were also higher in old yam tubers. Except for phosphorylase, these enzymes had alkaline pH optima.     

Starch Synthesis in Developing Potato Tubers

The activities of enzymes involved in starch metabolism were measured at intervals during tuberization and the early stages of tuber growth in Solanum tubersum grown in water culture under controlled environmental conditions. Starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase activities all increased during tuber development, the most pronounced increases occurring in the activities of ADP-glucose pyrophosphorylase and phosphorylase. The activity ratio ADPglucose pyrophosphorylase/phosphorylase was lowest in slow growing tubers and hightest in fast growing tubers. In addition, high sugar concentrations in fast growing tubers and low sugar concentrations in slow growing tubers suggested that enzyme levels might be influenced by sugar concentration. The activities of starch synthase, phosphorylase and ADPglucose pyrophosphorylase were increased 2–2.5 fold by the presence of 100 mM K⁺. It is concluded that the major enzyme changes occur as a consequence of tuber initiation and that starch accumulation is controlled, at least in part, by the activities of ADPglucose pyrophosphorylase and phosphorylase.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Yeast Glycogen Phosphorylase: Characterization of the Dimeric Form and Its Activation

The purification of yeast glycogen phosphorylase [EC 2.4.1.1] was improved by ethanol precipitation and affinity chromatography on a glycogen-Sepharose column. The purified enzyme gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a subunit molecular mass of 100 kDa. Gel electrophoresis also showed that the major activity of native phosphorylase was ascribed to a dimer of 203 kDa, which was agreed with the value obtained by gel filtration on Sephadex G-200. The yeast phosphorylase showed a high affinity for AMP- Sepharose, whereas the enzyme was specifically inhibited by AMP. This inhibition was competitive with respect to the substrate glucose 1-phosphate and gave a Ki value of 9.3 mm. Activation of the crude extract by phosphorylation with an endogenous phosphorylase kinase indicated that the yeast phosphorylase occurred in a mixture of phosphorylated and non-phosphorylated forms.     

山药异淀粉酶基因克隆及其在淀粉代谢中的作用北大核心CSCD

为了明确异淀粉酶基因(ISA 3)在山药淀粉代谢中的作用,该研究以‘毕克齐’和‘大和长芋’山药为试验材料,测定了块茎中淀粉及组分含量和异淀粉酶活性等;采用RT-PCR技术克隆了ISA 3,并进行生物学分析及山药块茎不同膨大期和不同组织间ISA 3基因的表达等。结果表明:(1)山药‘大和长芋’的直链淀粉、支链淀粉和总淀粉含量均显著高于‘毕克齐’,且两品种的淀粉含量随生长发育的变化均呈先升高后降低的趋势,并均于种植后120 d时达到最高,但‘毕克齐’的异淀粉酶(ISA)活性在整个膨大期均高于‘大和长芋’。(2)成功克隆获得山药ISA 3开放阅读框长1584 bp,编码527个氨基酸;ISA3为亲水性蛋白。(3)不同品种块茎在膨大时期的ISA 3基因表达趋势不同,‘毕克齐’中呈先显著上调随后下调,而在‘大和长芋’中表达总体下调,且在山药的叶、茎和块茎中均有表达,存在明显的组织特异性。(4)ISA活性与山药淀粉及支链淀粉含量呈显著和极显著正相关关系,但ISA活性与ISA 3的表达量呈负相关关系。研究表明,异淀粉酶参与了山药块茎中淀粉的合成,且主要对支链淀粉的合成起关键作用,ISA 3基因的表达可能对异淀粉酶活性和淀粉的合成起负调控作用。     

Glycogen phosphorylase from flight muscle of the hawk moth,Manduca sexta: purification and properties of three interconvertible forms and the effect of flight on their interconversion

Glycogen phosphorylase (EC 2.4.1.1) of Manduca sexta flight muscle was separated into three distinct peaks of activity on diethylaminoethyl-Sephacel. The three fractions of phosphorylase activity were further purified by affinity chromatography on AMP-Sepharose and shown to have the same relative molecular mass (=178000) on polyacrylamide gradient gel electrophoresis under non-denaturating conditions and to produce subunits of molecular mass =92000 on SDS gelelectrophoresis. On the basis of their kinetic properties with respect to the activator AMP and the inhibitor caffeine, the three fractions of phosphorylase activity were assigned as follows: peak 1=phosphorylase b (unphosphorylated form), peak 3=phosphorylase a (phosphorylated form); peak 2 represented a phospho-dephospho hybrid in which only one subunit of the dimeric enzyme was phosphorylated. This hypothesis was corroborated as the various forms could be interconverted in vitro by either dephosphorylation by an endogenous protein phosphatase producing the b form, or by phosphorylation catalyzed by purified phosphorylase kinase from rabbit muscle producing phosphorylase ab and a. From muscle of resting moths more phosphorylase was isolated in the b form (41%) than in the forms ab (28%) and a (31%), respectively. This proportion was changed in favour of the fully phosphorylated a form after a brief interval of flight when 68% of the phosphorylase activity was represented by the a form and only 13% by the b form. Unlike the phosphorylated forms a and ab of phosphorylase, the b form had low affinities for the substrates and for the activator AMP, and was virtually inactive if near-physiological concentrations of substrates and effectors were employed in the assays. The results demonstrate that in Manduca flight muscle three forms of phosphorylase coexist and that their interconversion is a mechanism for regulating phosphorylase activity in vivo.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EGTA ethyleneglycol-bis(-aminoethylether)N,N-tetra-acetic acid - M _r relative molecular mass - NMR nuclear magnetic resonance - PAGGE polyacrylamide gradient gel electrophoresis - P_i morganic phosphate - SDS sodium dodecylsulphate - TRIS tris(hydroxymethyl)-aminomethane - V _max maximum activity     

Trehalose phosphorylase from Pichia fermentans and its role in the metabolism of trehalose

During a screening for novel microbial trehalose phosphorylase three Pichia strains were identified as producers of this particular enzyme that have not yet been described. To our knowledge, this is the first time that this enzyme activity has been shown in yeasts. Pichia fermentans formed trehalose phosphorylase when cultivated on a growth medium containing easily metabolizable sugers such as glucose. Addition of NaCl (0.4 M) to the medium increased the synthesis of the enzyme significantly. Production of trehalose phosphorylase was found to be growth-associated with a maximum of activity formed at the transition of the exponential to the stationary phase of growth. Trehalose phosphorylase catalyzes the phosphorolytic cleavage of trehalose, yielding glucose 1-phosphate (glucose-1-P) and glucose as products. In vitro the enzyme readily catalyzes the reverse reaction, the synthesis of trehalose from glucose and glucose-1-P. For this reaction, the enzyme of P. fermentans was found to utilize -glucose-1-P preferentially. A partially purified enzyme preparation showed a pH optimum of 6.3 for the synthesis of trehalose. The enzyme was found to be rather unstable; it was easily inactivated by dilution unless Ca²⁺ or Mn²⁺ were added. This instability is presumably caused by dissociation of the enzyme. In contrast to other yeasts, P. fermentans rapidly degraded intracellularly accumulated trehalose when the carbon source in the medium was depleted. Trehalose phosphorylase seems to be a key enzyme in the degradative pathway of trehalose in P. fermentans. Additional enzymes in this catabolic pathway of trehalose include phosphoglucomutase, glucose-6-phosphate dehydrogenase, and gluconolactonase.This contribution is part of the Ph.D. thesis of Ingrid Schick     

Purification and some properties of polyphenol oxidase from the yam tubers, Dioscorea bulbifera

In a comparison of the polyphenol oxidase activity of various species of yam tubers the greatest enzyme activity was found in D. bulbifera. The enzyme was purified from acetone powder extracts of this plant. Ammonium sulphate fractionation, followed by ion exchange chromatography and gel filtration gave 22-fold purification. The final product gave a single band on polyacrylamide disc gel electrophoresis. The purified enzyme showed activity towards catechol, pyrogallol and dl-β-3,4-dihydroxyphenylalanine (dl-DOPA) and had a MW 115000 ± 2000. It was characterized by response to various inhibitors. β-Mercaptoethanol, dithioerythritol, l-cysteine, sodium metabisulphite and KCN inhibited strongly.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Partial purification and characterization of platelet factors stimulating the multiplication of normal human glial cells

Multiplication-stimulating activity for human glial cells was purified from human outdated platelets. By ion exchange chromatography anionic activity was separated from cationic activity. The former could be further separated by Sephadex G-200 gel chromatography into two peaks, whose molecular weights were 40 000 and < 10 000. The cationic activity was partially purified by concanavalin A (ConA) Sepharose chromatography, hydroxylapatite chromatography and SDS-polyacrylamide gel electrophoresis. The cationic activity was heterogeneous as demonstrated by isoelectric focusing (Ip 9.5–10.4), gel filtration on Bio-Gel P-150 and SDS-polyacrylamide gel electrophoresis (mol. wt 26 000–33 000). Less than 50 ng/ml was required of the factor to give a glial cell stimulation corresponding to that afforded by 1 % of human serum. A thymidine-degrading enzyme, present in human platelets and to a low degree also in human serum, was found to interfere with the assay for multiplication-stimulating activity. The enzyme (probably a thymidine phosphorylase) converted [³H]thymidine to [³H]thymine, causing a reduced incorporation of ³H into cellular DNA. This difficulty was circumvented by use of an autoradiographic estimation (per cent labelled nuclei) of the multiplication-stimulating activity.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Leupeptins Produced by the Marine Alteromonas sp. B-10–31

Endo-1,4-β-D-mannanase (1,4-β-D-mannanohydrolase, EC 3.2.1.78) was purified from viscera of a mud snail, Pomacea insularus (de Ordigny). The purified enzyme gave a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was estimated to be 44,000. The amino-terminal sequence was H· Gly-X-Leu-Arg-Arg-Gln-Gly-Thr-Asn-Ile-Val-Asp-Ser-His-Gly-His-Lys-Val-Phe-Leu-Ser-Gly-Ala-Asn-Thr-Ala-Trp-Val-Ala-Tyr-Gly-Tyr-Asp-. The enzyme was stable from pH about 5.0 to about 10.5 and had its maximum activity at pH about 5.5. The purified enzyme produced M2, M3, M4,and M5 from β-1,4-mannan. Enzyme activity was greatly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and dithiothreitol at 1 mM concentration. In addition, N-bromosuccinimide completely inhibited the enzyme activity.     

Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V _max of 2.03 × 10⁻⁹ s ⁻¹ and a K _m of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70°C and the enzyme had a half life at 60°C of 20.8 h.     

Pyruvate kinase,an enzyme subject to regulation inDioscorea alata

Different species of yam tubers were examined for the presence of pyruvate kinase and phosphatase activities, Pyruvate kinase was purified 25 fold with a yield of 50 %. using ammonium sulphate precipitation and ion exchange chromatography on DEAE-Sephadex. Partially purified enzyme showed normal Michaelis-Menten kinetics. However, pyruvate kinase from crude extract of dormant yam tuber showed slight sigmoid response towards phosphoenol-pyruvate and magnesium and to a certain extent ADP. The enzyme is activated by AMP and inhibited by ATP and citrate in both crude and partially purified preparations. Further studies on the effect of energy charge on the enzyme strongly suggest that pyruvate kinase from D. alata is a regulatory enzyme. No evidence was found for the presence of more than one pyruvate kinase in germinating D. alata tuber. With the exception of D. dumentorum, all the other three species of yams studied contain very little or no detectable phosphatase activity during dormancy. However, phosphatase activity increased during germination in all the species, thus excluding the use of sprouting yam tubers for kinetic study of pyruvate kinase.     

A New Peptide-N 4-(acetyl-β-glucosaminyl)asparagine Amidase from Soybean (Glycine max) Seeds: Purification and Substrate Specificity

We report here the isolation and characterization of a peptide-N ⁴-(acetyl-β-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the β-aspartylglycosylamine linkage (GlcNAcβ1→Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Biochemical Studies on Ricin Part X Effect of the Two Constituent Polypeptide Chains of Ricin D toward Mouse Sarcoma Ascite Tumor Cells

A maltotetraose-forming amylase from Pseudomonas stutzeri was highly purified by adsorption on starch granules and by chromatographies on Sephadex G-100 and DEAE-cellulose. The purified enzyme showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. The optimum pH for enzyme action on starch was 6.0-6.5, and the optimum temperature was 45°C. The purified enzyme attacked starch from the non-reducing end to produce α-anomer oligosaccharides. This indicated that the enzyme was an exo-α-amylase which had not hitherto been found. The enzyme activity was markedly inhibited by the addition of Cu²⁺, Hg²⁺, N-bromosuccinimide and 2,3-butanedione. The molecular weight of the enzyme determined by the method of Weber and Osborn was about 5.7 × 10⁴. The isoelectric point of the enzyme was estimated to be 5.3 by polyacrylamide gel electrofocusing. The Km and k₀ values of this enzyme for starch, glycogen, short chain amylose and some maltooligosaccharides were calculated from Lineweaver-Burk plots.     

Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds.

Rabbit brain purine nucleoside phosphorylase used in this study was purified 6000-fold to apparent homogeneity and a specific activity or 50 μmol min^?1 mg ^?1 protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound d,l-6-methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 × 10^?5M for folic acid and 3.80 × 10^?5m for d,l-6-methyl 5,6,7,8-tetrahydropterine. Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 × 10^?4 and 1.95 × 10^?4m. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory potency with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 × 10^?4m. The Michaelis constant for the substrate guanosine was near 6.0 × 10^?5m. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both histidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependent sensitivity of an essential catalytic group, and its probable location at the active site.