Anchimeric assistance in the intramolecular reaction of glucose-dehydrogenase-polyethylene glycol NAD conjugate

Polyethylene glycol-bound derivatives of NAD(P) (PEG-NAD(P)) are water-soluble macromolecular coenzymes used in continuous enzyme reactors. These NAD(P) derivatives have good coenzyme activity for many dehydrogenases, but some enzymes such as glucose dehydrogenase (EC 1.1.1.47) show very low activity with these derivatives (less than 0.1% of that for native NAD(P)). In this work, we prepared a covalently linked glucose-dehydrogenase-polyethylene glycol-NAD conjugate (GlcDH-PEG-NAD) and found that the conjugate shows a much higher reaction rate than that of the native enzyme plus PEG-NAD: the ratio of the reaction rates of GlcDH-PEG-NAD and the native enzyme plus PEG-NAD is calculated to be 10,000-fold at the concentrations of the enzyme subunit and NAD moiety of 0.31 and 0.65 microM, respectively; the rate of the conjugate is even higher than that of the native enzyme plus native NAD. This rate acceleration is due to the increase in the effective concentration of NAD moiety ("anchimeric assistance") and demonstrates the potential of covalent linking for improving the interaction between an enzyme and a coenzyme derivative.     

35-Cl nuclear magnetic resonance studies of anion-binding sites in proteins: lactate dehydrogenase.

³⁵Cl nmr relaxation rate measurements have been used to study anion-binding sites in pig heart lactate dehydrogenase. These studies reveal two types of sites, one is intimately associated with the active site, the other is not. The nonactive site has been ascribed to a subunit site in analogy with crystallographic results from the dogfish M₄ enzyme. The binding of either the reduced or the oxidized form of NAD results in an increase in the ³⁵Cl nmr relaxation rate by a factor of 1.8–2. The enhanced nmr relaxation rate of the binary lactate dehydrogenase-NAD complex is reduced on binding of the substrate inhibitor molecules oxamate or oxalate to a value less than that exhibited by lactate dehydrogenase alone. The enhancement of the nmr relaxation rate is attributed to a decrease in the dissociation constant of Cl for the enzyme. The K_p values for Cl binding to the active center site of lactate dehydrogenase is 0.85 m and for lactate dehydrogenase-NADH is 0.25 m. The ratio of these constants, 3.4, agrees well with the measured enhancement value 3.7. The effect of coenzyme analogs on the ³⁵Cl nmr relaxation rate has been examined. 3-Acetylpyridine NAD produces an enhancement of 4.3, thionicotinamide NAD of 2.3, whereas 3-pyridinealdehyde, adenosinediphosphoribose, and adenosine diphosphate do not affect the nmr relaxation state of Cl bound to lactate dehydrogenase.     

Alteration of Coenzyme Specificity of Lactate Dehydrogenase from Thermus thermophilus by Introducing the Loop Region of NADP(H)-Dependent Malate Dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k _cat?K _m ^coenzyme was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.     

Preparation and kinetic properties of 5-ethylphenazine-lactate-dehydrogenase-NAD+ conjugate, a semisynthetic lactate oxidase showing a hide-and-seek effect.

5-Ethylphenazine-lactate-dehydrogenase-NAD+ conjugate (EP(+)-LDH-NAD+) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min-1 and Km for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min-1 and Km for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD+ moieties of the conjugate are in the free state (i.e. not bound in the coenzyme-binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s-1 and 2.1 s-1 for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 microM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Prolindehydrogenase aus keimenden farnsporen

A soluble enzyme which converts proline to glutamic acid using NAD as coenzyme was isolated from young prothallia and spores of the fern Anemia phyllitidis. The purification was about 36-fold. The pH optimum is between 10·2 and 10·7; the K_m for proline is 4·6 × 10⁻⁴ M and for NAD 3·4 × 10⁻⁴ M. There are no multiple forms of this enzyme, as proved by gel electrophoresis.     

A novel caffeine dehydrogenase in Pseudomonas sp. strain CBB1 oxidizes caffeine to trimethyluric acid

A unique heterotrimeric caffeine dehydrogenase was purified from Pseudomonas sp. strain CBB1. This enzyme oxidized caffeine to trimethyluric acid stoichiometrically and hydrolytically, without producing hydrogen peroxide. The enzyme was not NAD(P)⁺ dependent; coenzyme Q₀ was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine.     

Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii

Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H₂ and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H₂ addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent K_m, V_max, and Q₁₀ values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H₂ production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.     

Purification,catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg²⁺ or Mn²⁺, with Mn²⁺ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K_m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k_cat was 69 s⁻¹. The half denaturationn temperature was 64°C, which was 20°C lower than that of the thermophile enzyme.     

Diphosphopyridine nucleotide-linked D-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus and the seaworm, Nereis virens. I. Physical and chemical properties

d-lactate dehydrogenase has been purified from horseshoe crab (Limulus polyphemus) skeletal muscle and the seaworm (Nereis virens). The purified Limulus dehydrogenase was shown to be a dimer, with a molecular weight of approximately 70 000. Sephadex gel filtration and equilibrium sedimentation yield molecular weights of about 80 000 and 70 000 respectively. Acid dissociation yields a molecular weight species of about 35 000. The native enzyme has an s^o₂₀^w of 3.95. Extrapolation of para-hydroxymercuribenzoate inhibition curves to 100% inhibition corresponds to two molecules of para-hydroxymercuribenzoate bound per molecule of enzyme. Studies on the stoichiometric binding of reduced coenzyme show two molecules bound per molecule of enzyme. The number of tryptic peptides has been found to be one-half that expected from the amino acid composition. The electrophoretic pattern of isoenzymic forms can be best interpreted as suggesting that the enzyme is dimeric. In vitro high salt, freeze-thaw hybridizations of the isolated Limulus muscle isoenzymes yield the electrophoretic pattern predicted by a dimeric structure.The physical properties ot Nereis lactate dehydrogenase have been found to be similar to those for the Limulus muscle lactate dehydrogenase.     

Covalent linking of poly(ethyleneglycol)-bound NAD with Thermus thermophilus malate dehydrogenase. NAD(H)-regeneration unit for a coupled second-enzyme reaction

Poly(ethyleneglycol)-bound NAD (PEG-NAD) was covalently linked to Thermus thermophilus malate dehydrogenase with a bifunctional reagent, 3,3'-(1,6-dioxo-1,6-hexanediyl)bis-2-thiazolidinethione. The covalently linked malate-dehydrogenase--PEG--NAD complex (MDH-PEG-NAD) was purified by DEAE-Sephadex column chromatography to remove unbound PEG-NAD, and fractionated by blue-Sepharose column chromatography into four preparations: MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 60-80% bound NAD moiety of these preparations of MDH-PEG-NAD was reduced by the enzyme moiety in the presence of L-malate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme. MDH-PEG-NAD I has the following properties. The Km value for exogenous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20-40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L-malate in the presence of the redox system of 5-ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s-1. The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH-PEG-NAD works as an NAD(H)-regeneration unit for coupled reactions.     

Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichiacoli

The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde: NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322. Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5′-AMP Sepharose, gel-filtration and ion-exchange chromatography. The amino acid composition was determined. The dehydrogenase was found to be a tetramer with identical 55 kDa subunits. Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred. The dehydrogenase was highly specific for betaine aldehyde. None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively. The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K⁺. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant.     

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway has been purified to 96% homogeneity. The native enzyme, which appears to be a tetramer, has an apparent molecular weight of 210000. The purified enzyme shows a narrow pH optimum at pH 7.8 and does not require ions for its catalytic activity. Under standard assay conditions the enzyme acts preferentially with NAD but reduces NADP at 11% of the rate observed for NAD, primarily because of a difference in K_m. Apparent K_m values are 6.4 μM for 5-carboxymethyl-2-hydroxymuconic semialdehyde and 52.2 μM for NAD.     

Characterization of a robust glucose 1-dehydrogenase,SyGDH, and its application in NADPH regeneration for the asymmetric reduction of haloketone by a carbonyl reductase in organic solvent/buffer system

To realize coenzyme regeneration in the reduction of haloketones, a codon-optimized gene Sygdh encoding glucose 1-dehydrogenase (SyGDH) was synthesized based on the putative GDH gene sequence (Ta0897) in Thermoplasma acidophilum genomic DNA, and expressed in E. coli BL21(DE3). Recombinant SyGDH was purified to homogeneity by affinity chromatography with the specific activity of 86.3 U/mg protein towards D-glucose at the optimum pH and temperature of 7.5 and 40 °C. It was highly stable in a pH range of 4.5–8.0 and at 60 °C or below, and resistant to various organic solvents. The K_m and catalytic efficiency (k_cat/K_m) of SyGDH towards NADP+ were 0.67 mM and 104.0 mM⁻¹ s⁻¹, respectively, while those towards NAD+ were 157.9 mM and 0.64 mM⁻¹ s⁻¹, suggesting that it preferred NADP+ as coenzyme to NAD+. Additionally, using whole cells of E. coli/Sygdh-Sys1, coexpressing SyGDH and carbonyl reductase (SyS1), as the biocatalyst, the asymmetric reduction of 60 mM m-chlorophenacyl chloride coupled with the regeneration of NADPH in situ was conducted in DMSO/phosphate buffer (2:8, v/v) system, producing (R)-2-chloro-1-(3-chlorophenyl)ethanol with over 99.9% ee_p and 99.2% yield. Similarly, the reduction of 40 mM α-bromoacetophenone in n-hexane/buffer (6:4, v/v) biphasic system produced (S)-2-bromo-1-phenylethanol with over 99.9% ee_p and 98.3% yield.     

The role of the nicotinamide and adenine subsites in the negative co-operativity of coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase.

The interaction of 3-aminopyridine-adenine dinucleotide, an NAD ⁺ 2 analogue which is fluorescent at the pyridine end of the molecule, with rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was investigated. The fluorescence properties of the AAD⁺ molecule were used to monitor the nicotinamide subsites ou the GPDHase tetramer, the fluorescent aminopyridine moiety of the molecule serving as an intrinsic probe. Although the binding of AAD⁺ wag found to be negatively co-operative, no conformational changes induced at the nicotinamide subsite upon coenzyme binding were found to be transmitted to neighboring subunits. These findings, in conjunction with our earlier findings and with the observation that different NAD⁺ analogues which differ in the chemistry of the pyridine moiety bind with different extents of co-operativity, enable us to offer specific roles for the nicotinamide and the adenine subsites in generating the negative co-operativity.It is suggested that the structure of the pyridine moiety of the coenzyme controls the mode of binding of the pyridine moiety to the nicotinamide subsite. This, in turn, controls the orientation of the adenine moiety with respect to its subsite, thereby determining the mode of the interactions between the adenine and its binding domain. As the propagation of conformational changes caused by these interactions to neighboring subunits is believed to be the cause of the negative co-operativity exhibited by this enzyme towards coenzyme binding, the structure of the pyridine moiety controls this phenomenon.     

Preparation of a NAD(H)-polymer matrix showing coenzyme function of the bound pyridine nucleotide

To a Sepharose gel the pyridine nucleotide NAD(H) has been bound using dicyclohexyl carbodiimide. In order to improve the steric availability of the nucleotide for added soluble enzymes such as dehydrogenases, a spacer molecule, ε-amino caproic acid, was inserted between the carbohydrate matrix and the nucleotide. The obtained preparation contained 56 μmoles NAD⁺/g dry polymer. The obtained matrix-bound NAD(H) was accepted as coenzyme by added lactate dehydrogenase. These preparations were still active after storage for several weeks at 4° C and could be used repeatedly without loss of activity. This represents the first necessary step taken in the preparation of compact closed systems consisting of “enzyme–coenzyme–coenzyme-regenerating enzyme” bound to individual polymer beads; such systems eliminate the need for continuous coenzyme addition.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Pig liver glyceraldehyde-3-P dehydrogenase: purification, crystallization, and characterization.

Glyceraldehyde 3-P dehydrogenase was purified approximately 250-fold from pig liver and crystallized. The purification procedure consisted of treating liver homogenates with zinc chloride, followed by ammonium sulfate fractionation and ion exchange chromatography. The enzyme was monodisperse in the ultracentrifuge with a sedimentation coefficient of s_20,w = 7.85 S. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single subunit band with an approximate molecular weight of 38,000. High-speed sedimentation equilibrium gave a molecular weight of 1.5 × 10⁵. Incubation of the enzyme with ATP at 0 °C caused a loss of its dehydrogenase activity; some of the lost activity was regained upon warming to room temperature. Sucrose density gradient studies of the ATP-treated enzyme revealed a decrease in its sedimentation coefficient from 7.8 to 3.85 S. In the forward reaction direction, the K_m for glyceraldehyde 3-P was 240 μm and the K_m for NAD was 12 μm. In the backward reaction direction, the K_m for NADH was 23 μm and the K_i for NAD was 850 μm. Pig liver glyceraldehyde-3-P dehydrogenase resembles the rabbit muscle enzyme in that it apparently contains 2 to 3 mol of tightly bound NAD. However, it differs strongly from that enzyme in its rate and extent of inactivation by ATP at 0 °C and by urea; the pig liver enzyme, like the yeast enzyme, dissociates much more slowly and much less completely than the rabbit muscle enzyme under comparable conditions.