Immobilization of the tetrameric and monomeric forms of pigeon liver malic enzyme on Sepharose beads

Pigeon liver malic enzyme was chemically attached to Sepharose 4B-CL beads. The enzyme lost approximately 50% of its original activity when immobilization was carried out with 5 mg CNBr/ml gel. Immobilization performed at pH 8.0 or pH 4.5 resulted in the formation of matrix-bound tetramer and monomer, respectively. Matrix-bound reconstituted tetramer was derived from matrix-bound monomer by mixing the latter with soluble enzyme at pH 4.5, then raised the pH of the solution to 8.0. The matrix-bound monomer was demonstrated to be enzymically fully active in terms of specific activity. The pH profile for the enzymic reaction was similar for both soluble and immobilized enzymes. However, the latter had a broader range for the optimum pH (pH 6.8-7.8). The Arrhenius plots for all immobilized enzyme forms were biphasic with inflection at approximately 27 degrees C. The apparent Michaelis constants for the substrates increased about 2-3-fold after immobilization. All immobilized enzyme forms, including the matrix-bound monomer, showed substrate inhibition at high concentrations of L-malate. Both high-affinity and low-affinity binding sites for Mn2+ existed for all immobilized enzyme forms. These results are consistent with an existing asymmetric model, but are not compatible with a sequential model for the enzyme tetramer. The immobilized enzyme was stable for at least four months at 4 degrees C. As compared to soluble enzyme, the immobilized enzyme was less inhibited by (NH4)2SO4 or NaCl. It was also resistant to inactivation with periodate-oxidized aminopyridine adenine dinucleotide phosphate, an affinity label for malic enzyme. Incubation of the immobilized enzyme (1.25 microM) with the reagent (5.6 mM) resulted in pseudo-first-order inactivation with a rate constant of 0.0108 min-1 that was at least an order of magnitude smaller than that for the soluble enzyme.     

Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+

As determined by EPR, malic enzyme from pigeon liver binds Mn2+ with a half-site stoichiometry of two tight binding sites (KD=6 to 10 mum) per enzyme tetramer and at two to four weak binding sites (KD=0.43 to 1.34 mM). The activation of malic enzyme by Mn2+ at high levels of L-malate shows biphasic kinetics yielding two activator constants for Mn2+. The dissociation constants of Mn2+ for both classes of sites are of the same order as the kinetically determined activator constants of Mn2+, indicating active site binding at both classes of binding sites. The binding of Mn2+ to the tight sites enhances the paramagnetic effect of Mn2+ on 1/T1 of water protons by a factor (epsilon) of 17, while binding at the weak sites yields a smaller epsilon of 11. The coenzymes TPN and TPNH have no effects on epsilon, while the carboxylic acid substrates L-malate and pyruvate and the inhibitors D-malate and oxalate significantly decrease epsilon. TPNH causes a 38-fold tightening of binding of the substrate L-malate to the enzyme-Mn2+ complex, consistent with the previously described highly ordered kinetic scheme, but only a 2-fold tightening of binding of the competitive inhibitor D-malate. The dissociation constant of L-malate from the quaternary E-Mn2+-TPNH-L-malate complex (32 muM) agrees with the Km of L-malate (25 muM), indicating active site binding. The dissociation constants of pyruvate from the ternary E-Mn2+-pyruvate complex (12 mM) and from the quaternary E-Mn2+-TPN-pyruvate complex (20 mM) are similar to the Km of pyruvate (5 mM), also indicating active site binding and a less highly ordered kinetic scheme for the reactions of pyruvate than for those of L-malate. Analysis of the frequency dependence of 1/T1 of water protons indicates that two fast exchanging water ligands remain coordinated to Mn2+ in the binary E-Mn2+ complex. The binding of the substrates L-malate and pyruvate and of the transition state analog oxalate to the E-Mn2+ complex decrease the number of fast exchanging water ligands on Mn2+ by approximately 1, but the binding of D-malate has no significant effect on this parameter, indicating the occlusion or replacement of a water ligand of the enzyme-bound Mn2+ by a properly oriented substituent on C-2 of the substrate. Occlusion rather than replacement of a water ligand by pyruvate is established by studies of 1/T1 of 13COO- and 13CO-enriched pyruvate which indicate second sphere Mn2+ to pyruvate distances of 4.6 A (COO-) and 4.8 A (CO) in the ternary enzyme-Mn2+-pyruvate complex. Formation of the quaternary complex with TPN increases these distances by 0.8 A, indicating the participation of a second sphere enzyme-Mn2+-(H2O)-pyruvate complex in catalysis. Thus, malic enzyme, like five other enzymes which utilize metals to polarize carbonyl groups, forms a second sphere complex with its substrate.     

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.     

Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.     

Inhibition and alternate-substrate studies on the mechanism of malic enzyme

A number of dead-end inhibitors and alternate substrates were examined to gain an understanding of the substrate specificity and mechanism of malic enzyme. Comparison of Ki values for competitive inhibitors suggested that binding of the l-carboxyl of L-malate is by ion pairing with lysine or arginine, while binding of the 4-carboxyl is weaker, and probably of the induced-dipolar type. The 2-hydroxyl hydrogen bonds to a catalytic group, which, when it is protonated, adsorbs the keto form of oxalacetate. Since the only molecule other than L-malate that is oxidized is L-malate-beta-amide, carbon 4 must be trigonal for substrate activity, although a tetrahedral carbon bearing one or two hydroxyl groups gives good binding. Hydroxy groups at carbon 3 contribute to binding, but prevent substrate activity. Hydroxy and ketomalonates are bound more strongly than any of the four carbon acids, suggesting that the latter are bound with some strain. In inhibition studies, pyruvate analogues were competitive vs. pyruvate but noncompetitive vs. malate, while malate analogues were competitive vs. malate and noncompetitive vs. pyruvate. These compounds thus bind to both enzyme-triphosphopyridine nucleotide (E-TPN) and enzyme-reduced triphosphopyridine nucleotide (E-TPNH), but only malate analogues prevent release of TPN, while pyruvate analogues prevent release of TPNH. Ketomalonate and oxalacetate, both of which are slowly reduced by the enzyme in the presence of TPNH and thus must combine in the keto form with E-TPNH,, appear to combine with E-TPN mainly in the gem-diol (or for oxalacetate, also the enol) form. The substrate for the decarboxylation of oxalacetate at pH 4.5 is the keto form.     

Identification of the catalytic triad residues of porcine liver acylamino acid-releasing enzyme

Acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] is a tetrameric serine protease, which belongs to the oligopeptidase family and specifically removes acetyl amino acids from N-terminally acetylated peptides. By using diisopropyl fluorophosphate, we previously identified one of the residues comprising the catalytic triad of this enzyme as Ser587 [Miyagi, M. et al. (1995) J. Biochem. 118, 771-779]. To elucidate the other two residues forming the catalytic triad of this new serine-type protease, wild-type and four mutant AAREs, in which each candidate residue of the catalytic triad deduced from sequence alignment with other oligopeptidases was substituted by site-directed mutagenesis, were expressed in Escherichia coli as fusion proteins with short peptide chains at both N- and C-termini of a subunit of porcine liver enzyme. All of the recombinant AAREs were estimated to have similar conformational and quaternary structures to the native porcine liver enzyme from their CD spectra and behavior on gel-filtration, but the mutants in which Ala587, Asn675, or Tyr707 was substituted for Ser587, Asp675, or His707, respectively, did not show detectable hydrolytic activity toward acetyl-L-methionyl L-alanine. These facts suggest that Ser587, Asp675, and His707 are essential residues for the AARE activity and comprise the catalytic triad of the enzyme in this order. Thus, AARE has been shown to have a protease-like domain in its C-terminal region, as do other proteins classified as members of the oligopeptidase family.     

Exploring a channel to the active site of copper/topaquinone-containing phenylethylamine oxidase by chemical modification and site-specific mutagenesis

Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.     

Identification of Asp95 as the site of succinimide formation in recombinant human glial cell line-derived neurotrophic factor

PURPOSE: To compare gadolinium-enhanced inversion-recovery magnetic resonance (MR) imaging with renal cortical scintigraphy in the diagnosis of childhood pyelonephritis. MATERIALS AND METHODS: Thirty-seven patients with fever-producing urinary tract infection underwent gadolinium-enhanced inversion-recovery MR imaging and technetium-99m renal cortical scintigraphy. Each study was read in double-blind fashion by two radiologists. The kidney was divided into three zones, and each was graded as positive, equivocal, or negative for pyelonephritis. RESULTS: Seventy kidneys (210 zones) were imaged. Twenty-six kidneys (54 zones) had evidence of pyelonephritis at both MR imaging and scintigraphy. Twenty-four kidneys (100 zones) were negative on both studies. Twelve kidneys (42 zones) were positive at MR imaging but negative at scintigraphy, and four kidneys (seven zones) were negative at MR imaging but positive at scintigraphy. The results of MR imaging for pyelonephritis were not equivalent to the results of scintigraphy (P = .001 for renal zones). The proportion of positive agreement between readers for the presence of pyelonephritis was 0.85 and 0.57 for MR imaging and scintigraphy, respectively. The proportion of negative agreement was 0.88 and 0.80 for MR imaging and scintigraphy, respectively. CONCLUSION: Gadolinium-enhanced inversion-recovery MR imaging enabled detection of more pyelonephritic lesions than did renal cortical scintigraphy and had superior interobserver agreement.     

Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis

Site-directed mutagenesis was used to identify the veratryl alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the mutants for veratryl alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by lignin peroxidase.     

Identification and characterization of cathepsin B as the cellular MARCKS cleaving enzyme

The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH4Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 x g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time- and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.     

Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis

Prolyl aminopeptidase (PAP) has been classified as a sulfhydryl enzyme on the basis of its high sensitivity to p-chloromercuribenzoate and heavy metals. Recently, however, the possibility of PAP being instead a serine enzyme has been raised as a result of two observations--the conservation of some residues among the PAPs hitherto sequenced, and a similarity to some serine hydrolases. This is the first report describing an attempt to identify the active residue by site-directed mutagenesis. The pap genes from Bacillus coagulans and Aeromonas sobria, were used for the study. The changes made were Cys62Ser and Ser101Ala for the first enzyme, and Thr92Ala and Ser146Ala for the second. For both enzymes, only the changes made on the serine residues resulted in their complete inactivation, indicating that PAP is a serine peptidase.     

Escherichia coli inorganic pyrophosphatase: site-directed mutagenesis of the metal binding sites

Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the k(app) value by approx. 2-3 orders of magnitude. At the same time, the Km values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild-type pyrophosphatase.     

Effect of prolonged starvation on the activities of malic enzyme and acetylcholinesterase in tissues of Japanese quail

During starvation muscle protein degradation is increased but the mechanism for this is uncertain. In this study Japanese quail were starved for 5 days and the activities of malic enzyme and acetylcholinesterase were determined in various tissues. SDS-polyacrylamide gel electrophoresis showed that the soluble proteins with molecular weights corresponding to 160, 120, 108, 99 and 38 kDa were absent in the liver of the starved group. In the pectoral muscle the soluble proteins with molecular weights corresponding to 69, 41 and 34 kDa were missing. The activity of malic enzyme in the liver, heart and pectoral muscle of the starved group decreased markedly whereas that of acetylcholinesterase increased markedly in the pectoral muscle (P < 0.005). It is concluded that in prolonged starvation acetylcholinesterase synthesis may be induced in tissues being subjected to protein catabolism and that this enzyme may be involved as a protease in protein degradation.     

Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL. Identification of Asp10 as the active site nucleophile by 18O incorporation experiments

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.     

Identification of a gene product induced by hard-surface contact of Colletotrichum gloeosporioides conidia as a ubiquitin-conjugating enzyme by yeast complementation

The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts. Chemical signals, such as the host's surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen Colletotrichum gloeosporioides only after the conidia are in contact with a hard surface. What role this contact plays is unknown. Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes, chip1 (Colletotrichum hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame (ubc1Cg) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to the Saccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. UBC1Cg can complement the proteolysis deficiency of the S. cerevisiae ubc4 ubc5 mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.