Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.     

Properties of a class C beta-lactamase from Serratia marcescens.

A beta-lactamase produced by a penicillin-resistant strain of Serratia marcescens was isolated and purified. The kcat. value for benzylpenicillin was about 5% of that observed for the best cephalosporin substrates. However, the low Km of the penam resulted in a high catalytic efficiency (kcat./Km) and the classification of the enzyme as a cephalosporinase might not be completely justified. It also exhibited a low but measurable activity against cefotaxime, cefuroxime, cefoxitin and moxalactam. Substrate-induced inactivation was observed both with a very good (cephalothin) or a very bad (moxalactam) substrate. The active site was labelled by beta-iodopenicillanate. Trypsin digestion produced a 19-residue active-site peptide whose sequence clearly allowed the classification of the enzyme as a class C beta-lactamase.     

Unique properties of purified, Escherichia coli-expressed constitutive cytochrome P4504A5.

Cytochromes P450 of the 4A family metabolize a variety of fatty acids, prostaglandins, and eicosanoids mainly at the terminal carbon (omega-hydroxylation) and, to a lesser extent, at the penultimate carbon [(omega-1)-hydroxylation]. In the present study, cytochrome P4504A5 (4A5) has been successfully expressed in Escherichia coli, with an average yield of enzyme of approximately 80 nmol/liter of cells. Spectroscopic characterization of the purified enzyme, using electron paramagnetic resonance and absolute and substrate-perturbed optical difference spectroscopy, showed that the heme of resting 4A5 is primarily low spin, but is converted primarily to high spin by substrate binding. The kcat and Km values for laurate omega-hydroxylation were 41 min-1 and 8.5 microM, respectively, in the absence of cytochrome b5, and 138 min-1 and 38 microM, respectively, in the presence of cytochrome b5. Hydroxylation of palmitate was dependent on the presence of cytochrome b5; kcat and Km values were 48 min-1 and 122 microM, respectively. Hydroxylation of arachidonic acid was barely detectable and was unchanged by the addition of cytochrome b5.     

OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.     

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.     

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.     

Purification and characterization of rat liver transglutaminase

1. Transglutaminase (EC 2.3.2.13) was purified from rat liver. 2. The enzyme was stable at 25 degrees C in the pH range of 6.0-9.0, with the optimum at pH 9.0. 3. The enzyme was inactivated after incubation for 20, 4 and 1 min at 44 degrees C, 52 degrees C, and 60 degrees C, respectively. 4. Activation energies were 30.4 kcal/mol for denaturation and 19.9 kcal/mol for substrate conversion to products. 5. The enzyme was inactivated by sulfhydryl modification with hydroxymercuribenzoate (99.1%) and N-ethylmalemide (78.5%). 6. Calcium, required for the activity, was replaced to a lesser extent, by Mg2+, Sr2+, Zn2+ and Mn2+ (31.8, 27.0, 24.6 and 3.5%). 7. Steady-state kinetics showed: Vmax = 10 microM-min-1, Km = 0.05 mM (N-dimethylated casein), kcat = 31.9 min-1 kcat/Km = 560 min-1 mM-1.     

Kinetic properties of triose-phosphate isomerase from Trypanosoma brucei brucei. A comparison with the rabbit muscle and yeast enzymes

The kinetic properties of Trypanosoma brucei brucei triose-phosphate isomerase are compared with those of the commercially available rabbit muscle and yeast enzymes and with published data on the chicken muscle enzyme. With glyceraldehyde 3-phosphate as substrate Km = 0.25 +/- 0.05 mM and kcat = 3.7 X 10(5) min-1. With dihydroxyacetone phosphate as substrate Km = 1.2 +/- 0.1 mM and kcat = 6.5 X 10(4) min-1. The pH dependence of Km and Vmax at 0.1 M ionic strength is in agreement with the results published for the yeast and chicken muscle enzymes. At ionic strength below 0.05 M the effect of a charged group specific for the trypanosomal enzyme and absent from the yeast and rabbit muscle enzymes becomes detectable. This effect significantly increases Km whereas Vmax becomes slightly higher. Trypanosomal triose-phosphate isomerase is inhibited by sulphate, phosphate and arsenate ions, by 2-phosphoglycolate and a number of documented inhibitors in the same concentration range as are the other triose-phosphate isomerases. The trypanocidal drug, Suramin inhibits T. brucei and rabbit muscle triose-phosphate isomerase to the same extent while leaving the yeast enzyme relatively unaffected.     

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Characterization of proline endopeptidase from rat brain

A homogeneous proline endopeptidase from rat brain is characterized with respect to its substrate specificity and the residues essential for catalysis. The two fluorogenic substrate analogues tested, pyroglutamylhistidylprolyl-beta-naphthylamide and pyroglutamy(N-benzylimidazolyl)-histidylprolyl-beta-naphthylamide, have higher Vmax values (19.5 and 26.9 mumol . min-1 . mg-1, respectively) and considerably lower Km values (0.034 and 0.020 mM, respectively) than pyroglutamylhistidylprolylamide (Vmax = 2.9 mumol . min-1 . mg-1 and Km = 4.1 mM). Both fluorogenic substrates give rise to pH optima and pH-rate profiles similar to those of the amide. Values of Km and kcat are determined as a function of pH. Km is pH independent, with the titration curve for kcatKm-1 implicating an active-site residue(s) with a pKa of 6.2. Proline endopeptidase can be completely inactivated by low concentrations of diisopropyl fluorophosphate with an observed second-order rate constant of 2.5 x 10(4) min-1 . M-1. The stoichiometry of the alkylphosphorylation is 0.83 mol/mol of enzyme. The pH dependence of the inactivation by diisopropylfluorophosphate implicates a residue(s) involved in covalent bond formation having a pKa of 6.0. These data suggest that proline endopeptidase is a serine proteinase.     

Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1

The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.     

PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.

FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.     

Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.     

Olefin oxygenation and N-dealkylation by dopamine beta-monooxygenase: catalysis and mechanism-based inhibition

In an initial communication [May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., & Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 110, 161-168], we reported that 1-phenyl-1-(aminomethyl)ethene hydrochloride (PAME) is an olefinic substrate for dopamine beta-monooxygenase (DBM; EC 1.14.17.1) which inactivates the enzyme in an apparent mechanism-based manner. The present study further characterizes this reaction. The inactivation reaction yields kinact = 0.23 min-1 at pH 5.0 and 37 degrees C and is strictly dependent on reductant (ascorbate) and oxygen. The DBM/PAME substrate reaction (apparent kcat = 14 s-1), shown to be stimulated by fumarate, gives the corresponding epoxide as product, identified by derivatization with 4-(p-nitrobenzyl)pyridine. However, the lack of DBM inhibition by alpha-methylstyrene oxide, and the observation of identical PAME/DBM inactivation rates in the absence and presence of preformed enzymatic PAME epoxide, indicates that free epoxide is not the inactivating species. A structure-activity study revealed that 4-hydroxylation of PAME (to give 4-HOPAME) increases both kinact (0.81 min-1) and apparent kcat (56 s-1) values, while 3-hydroxylation (to give 3-HOPAME) greatly diminishes inactivation activity while retaining substrate activity (apparent kcat = 47 s-1). 4-Hydroxy-alpha-methylstyrene was found to be a DBM inhibitor (kinact = 0.53 min-1) with weak substrate activity (apparent kcat = 0.71 s-1), while 3-hydroxy-alpha-methylstyrene and alpha-(cyanomethyl) styrene were found not to exhibit detectable DBM substrate activity and only weak inhibitory activity. 3-Phenylpropargylamine hydrochloride showed no detectable DBM substrate activity but rapidly inactivated the enzyme. A new substrate activity for DBM was discovered, N-dealkylation of N-phenylethylenediamine and N-methyl-N-phenylethylenediamine, and the lack of O-dealkylation activity with phenyl 2-aminoethyl ether and 4-hydroxyphenyl 2-aminoethyl ether indicates that DBM N-dealkylation proceeds via initial one-electron abstraction from the benzylic nitrogen heteroatom. With this new substrate and inhibitor reactivity information in hand, along with the other known substrate reactions, a DBM oxygenation mechanism analogous to that for cytochrome P-450 is proposed.     

Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

The kinetics of the activation of Glu- and Lys-plasminogen by single-chain urokinase (sc urokinase) derived from the transformed human kidney cell line TCL-598 have been studied and compared with two-chain urokinase (tc urokinase). Plasminogen activation was determined by the increase in fluorescence polarization of fluorescein-labeled aprotinin, a high affinity inhibitor of plasmin. This methodology allows plasmin generation by sc urokinase to be measured in functional isolation, with no interfering generation of tc urokinase, sc urokinase was found to activate plasminogen to plasmin with apparent Michaelis-Menten-type kinetics. The Km for Glu-plasminogen activation was 47.7 microM, with a catalytic constant of 2.91 min-1. Lys-plasminogen activation by sc urokinase was characterized by a Km of 11.7 microM and a kcat of 5.60 min-1. The Km values for the activation of Glu- and Lys-plasminogen by tc urokinase were found to be similar to those for activation by sc urokinase (36.8 and 9.0 microM, respectively), but the catalytic constants were higher at 36.0 and 118 min-1, respectively. Therefore, on the basis of the catalytic efficiency kcat/Km, sc urokinase seems to have 16-27-fold lower activity than tc urokinase. This activity of sc urokinase is in contrast to its lack of activity against a low molecular weight peptide substrate (less than 0.2% of the activity of sc urokinase). The activation of sc urokinase to tc urokinase by plasmin was also characterized (Km = 3.0 microM, kcat = 105 min-1). Using these data, it was possible to calculate the theoretical rate of plasminogen activation by sc urokinase in the absence of aprotinin, when tc urokinase is generated by the action of plasmin. The calculated rate was in good agreement with that determined experimentally using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide. These data demonstrate that sc urokinase has properties which distinguish it from conventional serine protease zymogens. The lack of activity against low molecular weight peptide substrates demonstrates the inaccessibility of the substrate-binding pocket. However, there is a moderate activity against plasminogen, suggesting that plasminogen may be acting as both an effector and a substrate for sc urokinase.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Preferential action of rat brain cathepsin B as a peptidyl dipeptidase converting pro-opioid oligopeptides

Purified rat brain cathepsin B (EC 3.4.22.1) converted prodynorphins or proenkephalins to shorter active forms by the preferential removal of C-terminal dipeptides. The substrate affinities for Met-enkephalin-Arg-Phe or -Arg-Gly-Leu were Km 46 and 117 microM, and kcat/Km ratios were 67 and 115 microM-1, min-1, respectively. Met-Enkephalin was inactivated by the same mechanism (Km-450 microM; kcat/Km = 0.12 microM-1 min-1). The comparison of cathepsin B hydrolysis for pro-opioids, a synthetic hexapeptide and its fragments, C-blocked peptides (pro-opioid amides, Met-enkephalin amide, substance P), and bovine myelin basic protein, provided information on the influence of the C-terminal residues on dipeptide release, the rates as correlated to peptide length, and the optimal arrangement of residues favoring scission at the P1-P'1 sites. The brain enzyme was stereospecific and did not act on peptides with C-terminal D-amino acid substituents. Arg hindered and Pro blocked the release of C-terminal dipeptides when in the P'2 positions. The suppression of dipeptide release by agents inhibiting endopeptidase actions such as E-64 and leupeptin, and the endogenous brain factor (cerebrocystatin) point to similar catalytic mechanisms for the exopeptidase action.     

Reverse reaction of smooth muscle myosin light chain kinase. Formation of ATP from phosphorylated light chain plus ADP

Incubation of smooth muscle phosphorylated heavy meromyosin in the presence of myosin light chain kinase, calmodulin, ADP, and Ca2+ results in a decrease of the protein-bound phosphate. The dephosphorylation is not due to phosphatase activity and is dependent on the presence of ADP and the active ternary myosin light chain kinase complex. Using 32P-labeled phosphorylated 20,000-dalton light chains as the phosphate donor, the formation of ATP from ADP can be demonstrated. This reaction requires the presence of Ca2+, calmodulin, and myosin light chain kinase. These results indicate that myosin light chain kinase can catalyze a reverse reaction and form ATP from ADP and phosphorylated substrate. The rate of the reverse reaction, kcat/KLC approximately 0.21 min-1 microM-1, is considerably slower than the forward reaction under similar conditions and is therefore detectable only at relatively high concentrations of myosin light chain kinase. For the reverse reaction, KmADP is approximately 30 microM and ATP is a competitive inhibitor, KIATP approximately 88 microM. For the forward reaction, measured with both isolated light chains and intact myosin, KmATP is approximately 100 microM and ADP is a competitive inhibitor, KiADP approximately 140 microM (myosin) and 120 microM (light chains). Thus, the affinity of ATP for the forward and reverse reactions is similar, but the affinity of ADP is higher for the reverse reaction. From the light chain dependence of the two reactions, the following was calculated: forward, Km = 5 microM, kcat = 1720 min-1, and reverse, Km = 130 microM, kcat = 27 min-1. In contrast to the data obtained with isolated light chains, it is suggested that, with intact myosin as substrate, the Km term is primarily responsible for determining the rate of the reverse reaction. With light chains phosphorylated at serine 19 and threonine 18, it was shown that both sites act as a phosphate donor, although the reverse reaction for threonine 18 is slower than that for serine 19.