Chemical modifications of ribonuclease U1.

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.     

Immobilization of RNase Rs via its carbohydrate moiety to aminoethyl-Bio-Gel P-2 and its application for the hydrolysis of RNA to 2′,3′ cyclic nucleotides

Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35–40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 μM periodate oxidized enzyme are allowed to react with 1 ml (packed volume) of AE-Bio-Gel P-2 at 6±1°C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K_m but resulted in a 2·5-fold decrease in the V_max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2′,3′ cyclic nucleotides in 14 h, at pH 5·5 and 37°C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.     

Chemical modification of ribonuclease T1 with ozone.

The single tryptophan residue in ribonuclease T1 [EC 3.1.4.8] was selectively oxidized by ozone to N'-formylkynurenine, which was then converted to kynurenine by acid-catalyzed deformylation in the frozen state. The two enzyme derivatives thus formed, NFK- and Kyn-RNase T1, lost enzymatic activity at pH 7.5, at which native RNase T1 most efficiently catalyzes the hydrolysis of RNA. At pH 4.75, the modified enzymes retained a decreased but distinct enzymatic activity toward RNA without alteration of substrate specificity, and Kyn-RNase T1 was four times more active than NFK-RNase T1. The binding of 3'-GMP to these modified enzymes decreased remarkably at pH 5.5, the optimum pH for binding to the intact enzyme. The gamma-carboxyl group of glutamic acid 58 was still reactive to iodoacetic acid after modification of tryptophan 59. The amounts of the carboxymethyl group introduced into NFK- and Kyn-RNase T1 were 0.36 and 0.59 mol, respectively, under conditions such that quantitative esterification of native RNase T1 takes place. CD spectroscopy indicated that the tertiary structure of the molecule was disordered in NFK-RNase T1, but not significantly in Kyn-RNase T1. It is concluded that tryptophan 59 functions in maintaining the active conformation of the protein structure, particularly in constructing the active environment for a functionally important set of groups involved in the binding of the substrate at the active site, although direct participation of in tryptophan the catalytic function of ribonuclease T1 is unlikely.     

Glycation of amino groups in protein. Studies on the specificity of modification of RNase by glucose

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.     

The preparation of CMP-sialic acids by using CMP-acylneuraminate synthase from frog liver immobilized on sepharose 4B.

A preparation of frog liver CMP-acylneuraminate synthase (2-10-fold enriched over the homogenate) obtained from DEAE-Sephadex A-50 chromatography of a 105,000g liver supernatant was bound to Sepharose 4B by the CNBr method. The enzyme retained 80-100% activity on binding and showed similar properties to the purified soluble enzyme from the same source with respect to Km, pH optimum and inhibition. The bound enzyme was stable to temperatures above 40 degrees C, in contrast with the soluble enzyme, and could be stored for 4 months at 2 degrees C with loss of 20% activity. The bound enzyme was used preparatively for the synthesis of radioactive and non-radioactive CMP-N-acetylneuraminic acid and CMP-N-glycolloylneuraminic acid. With suitable substrate concentrations and ratios, yields of 80% and over can be achieved.     

Effect of phosphate on the kinetics and specificity of glycation of protein

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffers in order to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues in or near the active site. In contrast, in the cationic buffers, 3-(N-morpholino)propane-sulfonic acid and 3-(N-tris(hydroxymethyl)methyl-amino)-2-hydroxypropanesulfonic acid, the kinetics of glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNAse was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increased glycation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effects on the glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establish that buffering ions or ligands can exert significant effects on the kinetics and specificity of glycation of proteins.     

Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred in the presence of 3'AMP. 1H-NMR titration and photo-chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and His115 were partially modified to yield a total of one mole of N tau-carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or His115 inactivates the enzyme.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2

RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.     

His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study.

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)     

Active site characterization of RNase Rs from Rhizopus stolonifer: involvement of histidine and lysine in catalysis and carboxylate in substrate binding.

Chemical modification studies on purified RNase Rs revealed the involvement of a single histidine, lysine and carboxylate residue in the catalytic activity of the enzyme. RNA could not protect the enzyme against DEP- and TNBS-mediated inactivation whereas, substrate protection was observed in case of EDAC-mediated inactivation of the enzyme. K(m) and k(cat) values of the partially inactivated enzyme samples suggested that while histidine and lysine are involved in catalysis, carboxylate is involved in substrate binding. Active site nature of RNase Rs suggests that the inability of the enzyme to readily convert 2',3'-cyclic nucleotides to 3'-mononucleotides is probably due to the absence of catalytically active second histidine residue.     

The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity.

The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild-type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site. Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme.     

Studies on N-acetylneuraminic acid aldolase

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37 degrees C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent K(i) for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive K(i) 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the in-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.     

Role of histidine-40 in ribonuclease T1 catalysis: three-dimensionalstructures of the partially active His40Lys mutant.

Histidine-40 is known to participate in phosphodiester transesterification catalyzed by the enzyme ribonuclease T1. A mutant enzyme with a lysine replacing the histidine-40 (His40Lys RNase T1) retains considerable catalytic activity [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. We report on the crystal structures of His40Lys RNase T1 containing a phosphate anion and a guanosine 2'-phosphate inhibitor in the active site, respectively. Similar to previously described structures, the phosphate-containing crystals are of space group P2(1)2(1)2(1), with one molecule per asymmetric unit (a = 48.27 A, b = 46.50 A, c = 41.14 A). The complex with 2'-GMP crystallized in the lower symmetry space group P2(1), with two molecules per asymmetric unit (a = 49.20 A, b = 48.19 A, c = 40.16 A, beta = 90.26). The crystal structures have been solved at 1.8- and 2.0-A resolution yielding R values of 14.5% and 16.0%, respectively. Comparison of these His40Lys structures with the corresponding wild-type structures, containing 2'-GMP [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368] and vanadate [Kostrewa, D., Hui-Woog Choe, Heinemann, U., & Saenger, W. (1989) Biochemistry 28, 7692-7600] in the active site, respectively, leads to the following conclusions. First, the His40Lys mutation causes no significant changes in the overall structure of RNase T1; second, the Lys40 side chains in the mutant structures occupy roughly the same space as His40 in the corresponding wild-type RNase T1 structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.     

Quantitation of the loss of the bacteriophage lambda receptor protein from the outer membrane of lipopolysaccharide-deficient strains of Escherichia coli.

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.     

Subunit interactions and immobilised dimers of human liver arginase.

Incubation of soluble human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) with p-hydroxymercuribenzoate resulted in the dissociation of the enzyme into active dimers. Addition of 2-mercaptoethanol resulted in the regeneration of the tetrameric enzyme. When arginase, bound covalently to nylon, was incubated with p-hydroxymercuribenzoate, matrix-bound dimers were obtained. Incubation of these species with 2-mercaptoethanol resulted in stable, unmodified dimers. Based on this dissociation of arginase, a model with D2-symmetry is suggested for this enzyme. The specific activity, the Km value for arginine, pH optimum and the inhibition constants for ornithine and lysine were determined for monomeric, dimeric and tetrameric forms. It is concluded that the behaviour of the active sites of the monomers is not substantially altered by the interaction of these species in the oligomeric molecule.     

Kinetic studies on soluble and insoluble urokinases.

A water-insoluble urokinase (ins-UK) was prepared by covalent coupling to an electrostatically neutral polyacrylamide derivative. The esteratic activity retained by the bound enzyme is about 70 percent of that of the soluble urokinase (UK). Comparative kinetic studies of these two forms of the enzyme were undertaken on lysine esters: N-alpha-acetyl-L-lysine-methyl ester (ALEe) and N-alpha acetylglycyl-L-lysine methyl ester (AGLMe). It was first observed that these substrates both exhibit a marked inhibitory effect toward soluble UK, whereas this phenomenon was less manifest with the insoluble form of the enzyme. Michaelis constants and maximal velocities measured at 33 degrees C, for UK and ins-UK, were identical when ALMe was used, but slightly different with AGLMe. Determination of initial velocities, at a series of pH values shows only minimal differences in the behavior of the soluble enzyme with respect to that of the insoluble form. However, over a range of temperatures, differing Km values for these two enzyme forms were obtained using AGLMe as the substrate. These last results suggest possible interactions between the substrate and the insoluble carrier of the enzyme.