The effects of pH and inhibitors upon the catalytic activity of the dihydroorotase of multienzymatic protein pyr1-3 from mouse Ehrlich ascites carcinoma

We have studied factors affecting the catalytic activity of dihydroorotase (EC 3.5.2.3), purified as part of a multienzymatic protein which contains carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase (ME pyr1-3) and which initiates de novo pyrimidine biosynthesis in mouse Ehrlich ascites carcinoma. The apparent Km value for N-carbamyl-L-aspartate increases by 2 orders of magnitude as the pH increases from 7.0 to 8.3, consistent with equilibration of dihydroorotase (E) between four states of protonation (E in equilibrium EH in equilibrium EH2 equilibrium EH3), where EH3 is the only catalytically active form of dihydroorotase for the biosynthetic reaction, having a Km for N-carbamyl-L-aspartate of 30 micro M. The apparent Km for L-dihydroorotate shows a converse dependence upon pH, remaining relatively constant at alkaline pH and increasing progressively as the pH is decreased below 7.0. These data are consistent with the above model if E and EH are catalytically active for the degradative reaction, both having Km values of 4.4 micro M for L-5,6-dihydroorotate. The D isomers of carbamylaspartate and dihydroorotate are also substrates for dihydroorotase. At pH 7.33, the apparent Km values for N-carbamyl-L-aspartate and N-carbamyl-D-aspartate are 247 and 204 micro M, respectively, but the Vmax for N-carbamyl-D-aspartate is only 1.7% of that obtained with N-carbamyl-L-aspartate. Orotate and a series of 5-substituted derivatives are competitive inhibitors of dihydroorotase. At pH 7.27, the apparent Ki for orotate using N-carbamyl-L-aspartate as substrate is 170 micro M and with L-5,6-dihydroorotate as substrate, the apparent Ki value is 9.6 micro M, suggesting that the enzyme exists in different forms in the presence of each substrate. Dihydroorotase is inhibited in a time-dependent manner by 50 mM L-cysteine and the presence of N-carbamyl-L-aspartate or L-5,6-dihydroorotate protects against this ultimately complete inactivation. 2-Mercaptoacetate, 2-mercaptoethylamine, 3-mercaptopropionate, and L-2,3-diaminopropionate have a similar although less potent inhibitory effect. To account for the data obtained, we propose a model for the equilibria existing between various protonated forms of dihydroorotase which is consistent with the pH dependencies of the apparent Km values observed and the Vmax values observed previously (Christopherson, R.I., and Jones, M.E. (1979) J. Biol. Chem. 254, 12506-12512). In addition, a catalytic mechanism is presented for the interconversion of N-carbamyl-L-aspartate and L-5,6-dihydroorotate.     

Purification of properties of dihydroorotase, a zinc-containing metalloenzyme in Clostridium oroticum.

Dihydroorotase +4,5-L-dihydro-orotate amidohydrolase [EC 3.5.2.3]), which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate, has been purified from orotate-grown Clostridium oroticum. The enzyme is homogeneous when subjected to polyacrylamide gel electrophoresis and is stable at pH 7.6 in 0.3 M NaCl containing 10 muM ZnSO4. The enzyme has a molecular weight of approximately 110,000. Sodium dodecyl sulfate gel electrophoresis, using three different buffer systems, indicated the enzyme is composed of two subunits, each having a molecular weight of 55,000. Dihydroorotase is shown by atomic absorption spectroscopy to be a zinc-containing metalloenzyme with 4 g-atoms of zinc per 110,000 g of protein. The pH optima for the conversion of N-carbamyl-L-aspartate to L-dihydroorotate and for L-dihydroorotate to N-carbamyl-L-aspartate are pH 6.0 and 8.2, respectively. The Km values for N-carbamyl-L-aspartate and for L-dihydroorotate are 0.13 and 0.07 mM, respectively. Inhibitor studies indicate that zinc may be involved in the catalytic activity of the enzyme.     

Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II).

Treatment of Escherichia coli dihydroorotase (a homodimer of subunit molecular weight 38,729) containing only the 1 active site Zn(II) ion per subunit with the sulfhydryl reagent N-(ethyl)-maleimide (NEM) blocks the two external Zn(II) sites per subunit and dramatically lessens the precipitation caused by high concentrations of Zn(II); stabilizes the enzyme partially against air oxidation and dilution inactivation; makes the active site Zn(II) easier to remove; and lowers Km and increases kcat. Treatment of NEM-blocked dihydroorotase ((NEM)dihydroorotase) with the chelator 2,6-pyridinedicarboxylic acid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(II) with a half-life of 15 min, allowing the production of milligram amounts of moderately stable apo-(NEM)dihydroorotase in about 80% yield. Treatment of apo-(NEM)dihydroorotase with Co(II) at pH 7.0 produces (NEM)dihydroorotase completely substituted at the active site with Co(II) in 100% yield: analysis gives 0.95-1.1 g atoms of Co(II) per active site and 0.03-0.05 g atoms of Zn(II) per active site. This Co(II)-(NEM)dihydroorotase is hyperactive at pH 8. The electronic absorption spectrum of Co(II)-(NEM)dihydroorotase at pH 6.5 implicates an active site thiol group as a ligand to the metal ion. The spectrum is inconsistent with tetrahedral coordination of the active site metal ion and is most consistent with a pentacoordinate structure.     

Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the mechanisms of CO2 hydration by native and Co2+-substituted carbonic anhydrase II

We have measured the pH dependence of kcat and kcat/Km for CO2 hydration catalyzed by both native Zn2+-and metallo-substituted Co2+-bovine carbonic anhydrase II in the absence of inhibitory ions. For the Zn2+-enzyme, the pKa values controlling kcat and kcat/Km profiles are similar, but for the Co2+-enzyme the values are about 0.6 pH units apart. Computer simulations of a metal-hydroxide mechanism of carbonic anhydrase suggest that the data for both native and Co2+-carbonic anhydrase can be accounted for by the same mechanism of action, if we postulate that the substitution of Co2+ for Zn2+ in the active site causes a separation of about 0.6 pH units in the pKa values of His-64 and the metal-bound water molecule. We have also measured the activation parameters for kcat and kcat/Km for Co2+-substituted carbonic anhydrase II-catalyzed CO2 hydration and have compared these values to those obtained previously for the native Zn2+-enzyme. For kcat and kcat/Km we obtain an enthalpy of activation of 4.4 +/- 0.6 and approximately 0 kcal mol-1, respectively. The corresponding entropies of activation are -18 +/- 2 and -27 +/- 2 cal mol-1 K-1.     

The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

Studies on Turbatrix aceti beta-N-acetylglucosaminidase. 2. Kinetic studies on the active site

The purified beta-N-acetylglucosaminidase isolated from Turbatrix aceti hydrolyzes both p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides. The enzyme had Km values of 0.28 and 0.23 mM, Vmax values of 104 and 69 mumol min-1 mg protein-1, and activation energies of 11.7 and 9.9 kcal/mol for the two substrates, respectively. Several lines of experimental evidence show that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same molecule at a single catalytic site. Substrate analogs were synthesized in which the acetamido group of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside, and their 1-thio analogs was modified by replacement of the amido-carbonyl oxygen with sulfur. These substrate analogs competitively inhibited both enzymatic activities. Analysis of the inhibition data indicates that a single catalytic site of the enzyme is responsible for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities. Competition kinetics between the two substrates further confirm the presence of a single active site for both activities. The pH dependence of the hydrolysis of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides has been determined. pKe1 and pKe2 values of 4.7 and 5.2, determined from the dependence of log Vmax/Km on pH, suggest that two carboxyl groups are involved in the reaction mechanism. The heats of ionization of the groups further confirm the above results.     

Evidence for a pH-dependent isomerization of Clostridium oroticum dihydroorotase

Analytical gel permeation chromatography on both Sephadex and polyacrylamide columns shows that Clostridium oroticum dihydroorotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) undergoes a large decrease in molecular size when the pH is decreased from 8 to 6. The Stokes radius decreases from about 40 A to 36 A. Neither the molecular size nor kinetic properties are dependent on protein concentration. Thus, the decreased molecular size reflects a pH dependent isomerization of the enzyme.     

The effect of Mn2+ and Co2+ on the activities of a zinc metallodipeptidase from a mouse ascites tumor.

Kinetic studies of the effect of addition of Co2+ or Mn2+ to a highly purified dipeptidase from Ehrlich-Lettré mouse ascites tumor cells show that these metals specifically activate the hydrolyses of certain classes of dipeptides. This enzyme was previously (S. Hayman and E. K. Patterson, 1971, J. Biol. Chem. 246, 660) reported to be a Zn-metalloenzyme. It is now shown that Zn is the only metal that can partially restore the activity of the EDTA-inhibited dipeptidase in cleaving Ala-Gly. Addition of Co2+ increases the Vmax of N-terminal Gly-dipeptides with increase in Km while addition of Mn2+ primarily activates the hydrolysis of Pro-Gly, again with increases in both Vmax and Km. Prior incubation (5 min, 30 degrees) of the dipeptidase with the appropriate metal ions causes decrease in initial lag time in the Co2+-activated hydrolysis of Gly-Gly and the Mn2+-activated hydrolysis of Pro-Gly. Long-term (6-19 hr, 0 degrees) incubation of the enzyme with Co2+ results in loss of activity toward Ala-Gly with a concomitant 13-fold increase in the rate of Gly-Gly hydrolysis and loss of 70% of the Zn2+ from the dipeptidase; these effects can be partially reversed by addition of Zn2+. In contrast, long-term incubation of the enzyme with Mn2+ results in no loss of Zn2+ and a twofold increase in activity toward Pro-Gly. One affinity constant of 1.4 muM for Co2+ and two constants of 0.23 and 27 muM for Mn2+ were determined by kinetic experiments. Comparison of the properties of this tumor enzyme with a dipeptidase purified in our laboratory from Escherichia coli B, and with mammalian dipeptidases highly purified by others, shows remarkable similarities in molecular weights and molecular activities toward the preferred substrates but in the case of bacterial dipeptidase, differences in substrate specificities and in the effect of metal ions.     

Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.     

Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis

Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two alpha (32 kDa) and two beta (28 kDa) polypeptides. This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from L-dihydroorotate to NAD(+), defines the C. oroticum enzyme as a family 1B dihydroorotate dehydrogenase. The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism. The pH-k(cat) profile is bell-shaped with a pK(a) of 6.4 +/- 0.1 for the ascending limb and 8. 9 +/- 0.1 for the descending limb; the pH-k(cat)/K(m) profile is similar but somewhat more complex. The pK(a) values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively. At saturating levels of NAD(+), the isotope effects on (D)V and (D)(V/K(DHO)), obtained upon deuteration at both the C(5)-proR and C(5)-proS positions of L-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH. At pH = 6.5, the magnitude of the double isotope effects (D)V and (D)(V/K(DHO)), obtained upon additional deuteration at C(6), is consistent with a mechanism in which C(5)-proS proton transfer and C(6)-hydride transfer occur in a single, partially rate-limiting step.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).     

Effect of membrane lipid environment on the activity of bovine adrenal 3-oxo-delta 5-steroid isomerase

The 3-oxo-delta 5-steroid isomerase (EC 5.3.3.1) activity from bovine adrenal cortex microsomes can be extracted in soluble form by the use of appropriate detergents, although recovery of enzyme activity is low (ca. 2%). Activity is restored upon removal of detergent and reconstitution of the enzyme into phospholipid vesicles. Both Km and Vmax of 3-oxo-delta 5-steroid isomerase of intact microsomes increase as the pH is raised from 7.5 to 9.5, with a particularly sharp increase (6- to 8-fold) above pH 8.5. The kinetic parameters of a detergent-solubilized isomerase preparation show little increase from pH 7.5 to 9.0, but isomerase reconstituted into artificial phospholipid vesicles demonstrates a 6- to 10-fold increase in both Km and Vmax over this pH range. Addition of Ca++ (1 mM) enhances the pH dependence of both Km and Vmax of the membrane-bound isomerase, causing a slight rise in Vmax/Km.     

Human placental dipeptidyl aminopeptidase III: hydrolysis of enkephalins and its stimulation by cobaltous ion

The degradation of enkephalin and related peptides by highly purified dipeptidyl aminopeptidase III (EC 3.4.14.4) was studied. The enzyme releases the N-terminal dipeptide units from substrates greater in length than the tetrapeptide. The enzyme exhibits an optimum of pH 7.5, Km of 81 microM and Vmax of 0.043 mumole/min for Leu-enkephalin. Its activity was markedly stimulated by Co2+, with both the Km and Vmax being increased. Among the enkephalin-related peptides examined, des-Tyr1-Leu-enkephalin was the most rapidly hydrolyzed with Co2+, but only slight stimulation was observed with Co2+.     

Alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium. Selective activation by manganese and effect of other divalent cations.

1. Alkaline p-nitrophenylphosphate phosphatase (pNPPase) activity of Halobacterium halobium is selectively stabilized and stimulated by Mn2+ ions. 2. Mn2+ binding to native pNPPase is characterized by a dissociation constant of 0.35 mM at pH 8.5, 37 degrees C, with a Hill coefficient of 0.988. 3. Mn2+ behaves as a mixed type nonessential activator, increasing the Vmax value (beta = 6.09, pH 8.5) and decreasing the Km value for pNPP (alpha = 0.56, pH 8.5). The Ki value for inorganic phosphate (a competitive inhibitor) was also decreased in the presence of Mn2+. 4. Activation of native pNPPase by preincubation with Mn2+ is a slow temperature-dependent process, which can be described by an exponential relationship vs time. However, a weak but immediate activation was also detected. 5. Zn2+, Cu2+ and Ni2+ were found to inhibit both native and Mn(2+)-stimulated pNPPase, whereas Co2+ and Cd2+ inhibited the Mn(2+)-stimulated pNPPase but had no effect on the native enzyme form.     

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(II)-containing enzyme

The essential Zn(II) in bovine liver dihydropyrimidine amidohydrolase (DHPase) was removed by incubation with 2,6-dipicolinic acid and replaced with Mn(II). Electron paramagnetic resonance studies of Mn(II) binding show that there are four binding sites per tetramer, and the dissociation constant at pH 7.5 is 13.5 microM. The substitution of Mn(II) for Zn(II) increases the specific activity of the enzyme approximately sixfold but has only a small effect (twofold increase) on the Km for 5-bromo-5,6-dihydrouracil (BrH2Ura). The pH dependence of the catalytic properties of Mn(II)-DHPase is the same as for the Zn(II) enzyme (Lee, M., Cowling, R., Sander, E., and Pettigrew, D. (1986) Arch. Biochem. Biophys. 248, 368-378). The pH dependence is well described in terms of the ionization of a single group with a pK of about 6 in the free enzyme. The ionization of this group is required for catalytic activity. The substitution of Mn(II) for Zn(II) does not affect the pH dependence of DHPase catalysis and therefore strongly suggests that the ionizable group is an amino acid residue at or near the active site, rather than a metal-bound water molecule. The pH dependence of the enhancement of the paramagnetic effect of the DHPase-Mn complex on the relaxation rate of the solvent water protons also is well described in terms of the ionization of a single group with a pK of about 6. Ionization of the group which is involved in catalysis also perturbs the environment of the bound Mn(II). The ionization of the active site group does not affect the number of exchangeable water molecules but does affect the symmetry of the environment of the bound Mn(II) and its electron relaxation.     

The pH-dependence of class B and class C beta-lactamases.

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.     

Purification and characterization of a pyridine nucleotide glycohydrolase from rabbit spleen

A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a Km of 0.25 mM, a Vmax of 5.3 mumol/min/mg protein, an activation energy of 7.9 kcal/mol, and a molecular weight of approximately 400,000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN:NAD:NADP = 1.00:1.45:0.44 and Vmax/Km ratios of 1.00:1.7:2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with Ki values similar to Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme in vivo.     

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k _cat of the Fe(III)Ni(II) derivative (approximately 60 s⁻¹) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the μ-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine.

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.