Characterization of porphobilinogen deaminase from rat liver

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.     

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).     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

Purification and kinetic studies on a porphobilinogen deaminase from the unicellular green alga Scenedesmus obliquus

Porphobilinogen deaminase, the enzyme condensing four molecules of porphobilinogen, was isolated and purified from light grown Scenedesmus obliquus (wild type). The purification procedure included heat treatment, ammonium sulphate fractionation, gel filtration, high-resolution anion-exchange chromatography and hydrophobic interaction chromatography. The enzyme was purified 1368-fold, compared to the initial crude extract. Its final specific activity was 6812 units · (mg · protein)^?1 at pH 7.4 with a recovery of 44%. The relative molecular mass was 33000, as determined by Sephadex G-100 gel filtration, and 35900 by lithium dodecyl sulfate-polyacrylamide-gel electrophoresis, indicating that the enzyme is a monomer. Studies of initial reaction velocities showed a linear progress curve for hydroxymethylbilane formation and a hyperbolic dependence of the initial reaction rate on substrate concentration, consistent with a sequential displacement mechanism. Apparent kinetic constants (K _m and V _max) for the conversion of porphobilinogen to hydroxymethylbilane at 37 ° C, pH 7.4, were 79 μM and 176 pmol · min^?1, respectively. Variation of both V _max and K _max with pH indicated the presence of ionizable groups in the enzyme-substrate complex(es), showing a single ionization (pK 7.15) in V _max/K _m plots. A sharp pH-profile for V _max was interpreted as a positive cooperative proton dissociation. In spite of the two pathways existing for 5-aminolevulinate biosynthesis in Scenedesmus, currently there is no indication of the existence of two porphobilinogen deaminases or even of isoenzymes.     

Zwitterionic and anionic forms of a serotonin analog as transport substrates

4,6-Difluoroserotonin, a serotonin analog with an acidic 5-hydroxyl proton (pK alpha = 7.97) relative to serotonin (pK alpha = 10.73), was tested as a substrate for the biogenic amine transporter of bovine chromaffin granules and the plasma membrane serotonin transporter of human blood platelets. The platelet serotonin transporter transports this analog with identical rates as those for serotonin, both at pH 6.7, where the hydroxyl group is predominantly protonated and at pH 9, where it is largely dissociated. In contrast, the chromaffin granule biogenic amine transporter prefers the form of 4,6-difluoroserotonin with a protonated 5-hydroxyl group. Thus, the KM for 4,6-difluoroserotonin increases, and Vmax decreases (relative to the values for serotonin) as the pH increases from 7 to 9. This effect may reflect a specific requirement for the protonated hydroxyl group in substrate translocation, as opposed to binding, since the KI for 4,6-difluoroserotonin inhibition of serotonin transport is the same as the KM for serotonin from pH 7 to 9.     

Transglucosidic reactions of the Aspergillus niger family 3 beta-glucosidase: qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH

The hydrolytic and transglucosidic reactions of the Aspergillus niger Family 3 beta-glucosidase were characterized. Michaelis-Menten plots of the rates of aglycone formation were normal (hyperbolic) at low [substrate]. However, at high [substrate] the rates decreased at pH below approximately 5.5 but increased at pH above approximately 5.5. Each decrease or increase took the form of a second hyperbola adjoining the first. Thin layer chromatography, gas-liquid chromatography, and NMR analyses indicated that the substrates became transglucosidic acceptors when present at high concentrations. When pNPGlc and cellobiose reacted as acceptors, the C6 hydroxyl of the non-reducing substrate component reacted to form beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-p-nitrophenol and beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-(1-4)-D-glucopyranose, respectively. The acceptor action accounted for the second adjoining hyperbolas. Rate equations were derived for the production of the aglycone and the transglucosidic intermediate, and these equations described the data very well. Hydrolytic Vmax {Vmax(h)}, hydrolytic Km {Km(h)}, transglucosidic Vmax {Vmax(t)}, and transglucosidic Km {Km(t)} values were obtained by non-linear regression analysis using these equations. Vmax(h) pH profiles were bell shaped with optima between pH 4 and 4.5 but the Vmax(t) values did not change substantially between pH 3 and 7. These differences in the pH profiles explain the decreasing and increasing adjoining hyperbolas since Vmax(t) is lower than Vmax(h) at pH less than approximately 5.5 but higher than Vmax(h) at pH greater than approximately 5.5. The reason for these pH effects is that the value of the hydrolytic rate constant (k3) decreases while the value of the transglucosidic rate constant (k4) does not change between pH 3 and 7. The study also showed that gentiobiose forms by an intermolecular reaction of the C6 hydroxyl of Glc rather than an intramolecular reaction and that an equatorial orientation of the C2 hydroxyl, the presence of a C6 primary hydroxyl and beta-linkages with oligosaccharide acceptors are important for acceptor reactivity.     

Variable H+/substrate stoicheiometries in Rhodotorula gracilis are caused by a pH-dependent protonation of the carrier(s).

Two carrier-mediated systems transport sugars in the yeast Rhodotorula gracilis depending on the pH. One system, with higher affinity for sugars, catalyses a symport of protons with sugar, whereas the other system, having lower affinity, is independent of protons. This was shown in three different ways. (1) At low pH, where only the high-affinity system works, a H+/sugar stoicheiometry of 1 was found. An increase of the pH and of the sugar concentration, which allowed the low-affinity system to operate, brought about a drop of the stoicheiometry to values below 1. (2) During H+ symport the influx of positive charge was electrically compensated by an equivalent efflux of K+ from the cells. At high pH and high sugar concentrations this stoicheiometry of K+ and sugar decreased concomitant with the H+/sugar stoicheiometry. (3) At pH 7.5 both transport systems were operating, as shown by biphasic saturation kinetics. Under these conditions only the high-affinity transport was found to be electrogenic. These results agree with the theory of an electrogenic H+/sugar symport where changes in the affinity for substrate are brought about by reversible protonation and deprotonation of the carrier.     

Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity

Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.     

Characterization of the multiple forms of hydroxymethylbilane synthase from rat spleen.

Phenylhydrazine treatment induced hydroxymethylbilane synthase activity (EC 4.3.1.8) in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulphonyl fluoride only three forms, two major and one minor, were resolved by the fractionation, suggesting that two of the original forms arose by proteolytic modification. Heat treatment (70 degrees C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes from rat spleen and erythrocytes suggested that the enzyme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr was found to be 43000 +/- 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. For the heat-stable form Vmax, varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.     

Porphobilinogen deaminase is unstable in the absence of its substrate

Porphobilinogen deaminase is induced during the dimethyl sulfoxide-mediated differentiation of Friend erythroleukemia cells. We have previously shown that when succinylacetone, a potent inhibitor of porphobilinogen formation, is present during the differentiation process, the induction of the enzyme is apparently suppressed. Here, we provide evidence that, in this condition, porphobilinogen deaminase is synthesized normally but does not accumulate as a consequence of an accelerated turnover. The normal half-life of the protein is 24 h but decreases to 10 h when the formation of its substrate is impaired by succinylacetone. We propose that when the enzyme is covalently bound to its substrate, a normal step in this enzymatic reaction, it is protected from proteolytic degradation, and we show that this new finding is relevant to the human disorder acute intermittent porphyria.     

Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase

This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.     

The pH and temperature dependence of the activity of the high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The hydrolysis of adenosine 3':5'-monophosphate by the high Km cyclic nucleotide phosphodiesterase of bakers' yeast was studied over a range of temperature and pH at I = 0.17. The effects of ionic strength and MgCl2 concentration were studied at pH 7.7 and 30 degrees C. Km and Vmax were insensitive to changes in the MgCl2 concentration between 1 and 30 mM, implying that this enzyme (which does not require free divalent metal ions) does not discriminate between free cyclic AMP- and the Mg-cyclic AMP+ complex. Vmax decreased below pH 6.8 because of protonation of a group required in the basic form in the enzyme x substrate complex. On the basis of its pK (5.46 at 30 degrees C) and delta H (23 kJ/mol) this group was tentatively identified as imidazole. Vmax/Km decreased above pH 6.8 because of ionization of a group required in the acid form in the free enzyme, with a pK of 7.88 at 30 degrees C and a delta H of about 13 kJ/mol. Several possibilities exist for the identity of this group, the most likely being a second imidazole, sulfhydryl, or a water molecule bonded to tightly bound zinc. At pH 7.90, log Vmax and log Km both changed linearly with 1/T (between 12 degrees C and 37 degrees C) with enthalpies of 47 and 55 kJ/mol, respectively. Consequently, at low enough cyclic AMP concentration, the rate of reaction at pH 7.90 decreases slightly when the temperature is increased. This is also true at higher pH, but in the physiological pH range (6.4 to 7.5) Vmax/Km and, therefore, the rate of reaction at very low cyclic AMP concentration were nearly independent of temperature. Under physiological conditions, the Km approaches the upper limit of in vivo cyclic AMP concentrations in yeast, and at normal in vivo cyclic AMP concentrations the pH optimum is within or below the physiological range of pH in yeast.     

Mechanism of modulation of dopamine beta-monooxygenase by pH and fumarate as deduced from initial rate and primary deuterium isotope effect studies

Dopamine beta-monooxygenase catalyzes a reaction in which 2 mol of protons are consumed for each turnover of substrate. Studies of the pH dependence of initial rate parameters (Vmax and Vmax/Km) and their primary hydrogen isotope effects show that at least two ionizable residues are involved in catalysis. One residue (B1, pK = 5.6-5.8) must be protonated prior to the carbon-hydrogen bond cleavage step, implying a role for general-acid catalysis in substrate activation. A second protonated residue (B2, pK = 5.2-5.4) facilitates, but is not required for, product release. Recent measurement of the intrinsic isotope effect for dopamine beta-monoxygenase [Miller, S. M., & Klinman, J. P. (1983) Biochemistry (preceding paper in this issue)] allows an analysis of the pH dependence of rate constant ratios and in selected instances individual rate constants. We demonstrate large changes in the rate-determining step as well as an unprecedented inversion in the kinetic order of substrate release from ternary complex over an interval of 2 pH units. Previously, fumarate has been used in dopamine beta-monooxygenase assays because of its property of enzyme activation. Studies of the pH behavior in the presence of saturating concentrations of fumarate have shown two causes of the activation: (i) fumarate perturbs the pK of B1 to pK = 6.6-6.8 such that the residue remains protonated and the enzyme optimally active over a wider pH range; (ii) fumarate decreases the rate of dopamine release from the ternary enzyme-substrate complex, increasing the equilibrium association constant for dopamine binding. Both effects are consistent with a simple electrostatic stabilization of bound cationic charges by the dianionic form of fumarate.     

Effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase on the pKa values of active site residues and on the pH dependence of catalysis.

The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D (15)N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK(a) value of 7.1 could result most simply from the averaging of two limiting pK(a) values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the beta-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK(a) value to 8.2. In the R39A mutant, the kinetically determined pK(a) of Pro-1 was also low, 5.0 +/- 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 +/- 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.     

The mechanism of hydrolysis of beta-glycerophosphate by kidney alkaline phosphatase.

1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector.     

Effects of strong electrolyte upon the activity of Clostridium perfringens sialidase toward sialyllactose and sialoglycolipids.

Clostridium perfringens sialidase was purified by affinity chromatography. Kinetic properties of the enzyme were examined with sialyllactose and with mixed sialoglycolipids (gangliosides) as substrates. With the latter substrate in 0.01 M Tris-acete in the absence of strong electrolyte, the pH optimum for enzymatic activity was 6.8. Addition of strong electrolyte (0.01 to 0.10 M Nac1) to the reaction medium caused an acidic shift and a broadening of the pH optimum, Enzymatic activity at pH 5.8 rose approximately 2.5-fold; a concomitant loss of activity at pH 6.8 was also observed. The alteration of enzymatic activity caused by strong electrolyte were dependent upon changes in Vmax. Km remained nearly invariant. Thus, a reversible transition of the enzyme from a relatively inactive to a highly active form occurred as a function of strong electrolyte concentration. Determination of the pK values of the active functional groups of C. perfringens sialidase revealed that the effects of strong electrolyte were exerted upon the pKa group of the enzyme. Strong electrolyte appeared to shield unfavorable electrostatic interactions between polyanionic sialoglycolipid micelles and the enzyme molecule, thus protecting the pKa group from inactivation. In comparision with the effects of strong electrolyte upon enzymatic activity toward the sialoglycolipid substrate, those observed with the monovalent substrate, sialyllacthose, were minor. Collectively, these findings indicate that ionic environment may effectively control the activity and relative substrate specificity of C. perfringens sialidase at a given pH. Furthermore, they explain the low pH optima and skewed pH profiles previously reported for enzymatic activity toward high molecular weight substrates.     

pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea).

The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 x 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 x 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.     

Pyrrolooxygenase isoenzymes from wheat germ

Both porphobilinogen oxygenase and skatole pyrrolooxygenase of wheat germ have isoenzyme forms of different charge. The more cationic isoenzymes were eluted from DEAE-cellulose with 10 mM Tris-HCl buffer (pH 7.6) and the less cationic were eluted with 50 mM NACl in the same buffer. The former had almost twice as many free amino groups (per mg of protein) as the latter. The more cationic isoenzyme was more sensitive to chelating agents and to acid treatment. They were differently inhibited by sodium dodecyl treatment and by temperature inactivation. Porphobilinogen oxygenase isoenzymes showed different activities with different buffers and also differed in their kinetics.     

Pyruvate and ketone-body transport across the mitochondrial membrane. Exchange properties, pH-dependence and mechanism of the carrier.

The effects of exchangeable ions and pH on the efflux of pyruvate from preloaded mitochondria are reported. Efflux obeys first-order kinetics, and the stimulation of efflux by exchangeable ions such as acetoacetate and lactate obeys Michaelis--Menten kinetics. The apparent Km value +/- S.E. for acetoacetate was 0.56 +/- 0.14 mM (n = 5) and that for lactate 12.3 +/- 2.3 mM (n = 6). The Vmax. values +/- S.E. at 0 degrees C were 16.2 +/- 2.0 and 21.9 +/- 2.7 nmol/min per mg of protein. The exchange of a variety of other substituted monocarboxylates was also studied. Efflux was also stimulated by increasing the external pH. The data gave a pK for the transport process of 8.35 and a Vmax. of 3.31 +/- 0.14 nmol/min per mg. The similarity of the Vmax. values for various exchangeable ions but the difference of this from the Vmax. in the absence of exchangeable ions may indicate that transport of pyruvate occurs with H+ and not in exchange for an OH- ion. The inhibition of transport by alpha-cyano-4-hydroxycinnamate took several seconds to reach completion at 0 degrees C. It is proposed that inhibition occurs by binding to the substrate site and subsequent reaction with an -SH group on the inside of the membrane. The inhibitor can be displaced by substrates that can also enter the mitochondria independently of the carrier and so compete with the inhibitor for the substrate-binding site on the inside of the membrane. A mechanism for transport is proposed that invokes a transition state of pyruvate involving addition of an -SH group to the 2-carbon of pyruvate. Evidence is presented that suggests that ketone bodies may cross the mitochondrial membrane either on the carrier or by free diffusion. The physiological involvement of the carrier in ketone-body metabolism is discussed. The role of ketone bodies and pH in the physiological regulation of pyruvate transport is considered.