Mole Of Enzyme Research Articles

Effectors of HIV-1 Protease Peptidolytic Activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.

Enzyme activities in the light of the membrane hypothesis of aging: [An answer to K. Kitani, Mech. Ageing Dev. 107 (1999) 299–322

The paper of Kitani cited in the title has raised an apparent contradiction regarding the validity of certain aspects of the membrane hypothesis of aging (MHA). He collected data showing that a number of detoxifying liver enzyme activities, although decline with age in male Fischer 344 rats, remain at an unchanged level in females of the same strain. He concluded that the main assumption of the MHA, according to which intracellular enzyme activities generally decline with age, cannot be maintained, and invoked me (p. 312) ‘… to provide in the future ample (and convincing) evidence’ in this respect. The present paper answers this criticism by showing that the apparent contradiction mentioned above is based on a misunderstanding on behalf of Kitani. Namely, MHA speaks about the general, density-dependent decline of the catalytic rate constant of any enzyme ( k cat), i.e., activity per mole of enzyme, being the true specific activity of the enzymes. This parameter inevitably decreases at the increased physical density of the intracellular colloids during aging. This statement derives from the molecular enzyme kinetic models, and has extensively been proven experimentally, too. On the other hand, Kitani speaks about enzyme activities per mg total protein content of certain tissue extracts, which is a very illdefined parameter, since the concentration of the measured enzyme remains unknown. Therefore, this latter parameter is irrelevant from the point of view of MHA in any aspect.

An inducible 1-butanol dehydrogenase, a quinohaemoprotein, is involved in the oxidation of butane by "Pseudomonas butanovora".

Butane-grown "Pseudomonas butanovora" expressed two soluble alcohol dehydrogenases (ADHs), an NAD(+)-dependent secondary ADH and an NAD(+)-independent primary ADH. Two additional NAD(+)-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD(+)-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway. BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1.0 mol pyrroloquinoline quinone (PQQ) and 0.25 mol haem c as prosthetic groups. BDH was synthesized as a monomer of approximately 66 kDa. It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C(4) diols and aromatic alcohols. It exhibited the lowest K:(m) (7+/-1 microM) and highest k(cat)/K:(m) (72x10(4) M(-1) s(-1)) value towards 1-butanol. BDH exhibited ferricyanide-dependent ADH activity. Calcium ions (up to 10 mM) increased BDH activity substantially. Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni. The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown "P. butanovora".

Analysis of conformational states of Candida rugosa lipase in solution: Implications for mechanism of interfacial activation and separation of open and closed forms

In this study, an analysis of the transition between the inactive (“closed”) and active (“open”) conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphates. It is shown that >90% inhibition of the enzyme activity toward water-soluble substrates (esterolytic activity) can be achieved with as little as 0.3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsified substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, which even exceeds that of the untreated control. These experimental observations suggest that the inhibitors interact predominantly, if not exclusively, with the open form of the enzyme and that any transitions occurring between the two conformers of the enzyme in solution, in the absence of an interface, are extremely slow. This conclusion is verified by separating the open and closed forms of the enzyme by hydrophobic interaction column chromatography on phenyl-sepharose. Fractions enriched with the respective conformations of the enzyme are further purified using gel-permeation chromatography. On the basis of the elution pattern from this step, and sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in the absence of interface) form of the lipase is found to be present in solution as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triolein. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 108–118, 2001.

The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

In an effort to prepare Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia for future spectroscopic and mechanistic studies, two methods for the preparation of Co(II)-L1 were tested. Method A involved adding CoCl2 directly to apo-L1 under anaerobic conditions. The resulting enzyme contained 1.9 moles of cobalt and exhibited very little activity, and UV-Vis, 1H NMR, and EPR studies indicated that most of the cobalt in this sample was Co(III). Method B involved reducing the single and unique disulfide bridge in L1 with tris(carboxyethyl)phosphine prior to adding CoCl2. The resulting enzyme was pink, contained 2.5 moles of cobalt per mole of enzyme, and exhibited kcat and Km values of 18+1 s(-1) and 10+/-1 microM, respectively, when using nitrocefin as the substrate. UV-Vis, 1H NMR, and EPR studies revealed that this enzyme sample contained high-spin Co(II). The UV-Vis spectra also provided evidence for Co(II) bound to one or both of the reduced cysteines. Efforts to block this non-specific Co(II) binding site using a chemical modification agent or site-directed mutagenesis were unsuccessful. The data presented here demonstrate the problem of solvent-exposed disulfides when preparing Co(II)-substituted enzymes and offers a convenient method to circumvent the problem.

Inactivation of cytochrome P450 2B1 by benzyl isothiocyanate, a chemopreventative agent from cruciferous vegetables.

A series of arylalkyl isothiocyanates were evaluated for their ability to inactivate purified cytochrome P450 2B1 in a reconstituted system. Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibition of cytochrome P450 (P450) enzymes has been implicated in their chemopreventative abilities. The naturally occurring isothiocyanates BITC and PEITC inactivated P450 2B1 in a time- and concentration-dependent manner, whereas the synthetic isothiocyanates phenylpropyl and phenylhexyl isothiocyanate did not result in inactivation, but were potent competitive inhibitors of P450 2B1 activity. The kinetics of inactivation of P450 2B1 by BITC were characterized. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivated in a mechanism-based manner. The loss of O-deethylation activity followed pseudo-first-order kinetics, was saturable, and required NADPH. The BITC concentration required for half-maximal inactivation (K(I)) was 5.8 microM, and the maximal rate constant for inactivation was 0.66 min(-)(1) at 23 degrees C. BITC was a very efficient inactivator of P450 2B1 with a partition ratio of approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was also investigated. More than 80% of the catalytic activity was lost within 12 min with a concomitant loss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude of the UV/visible absorption spectrum of the inactivated protein did not decrease significantly, and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC and protein precipitation analyses indicated that the P450 apoprotein was covalently modified by a metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 +/- 0. 16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggesting the modification of a single amino acid residue per molecule of enzyme that was inactivated. The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1 and that inactivation occurs primarily through protein modification.

Modulation of the Catalytic Activity of the Src Family Tyrosine Kinase Hck by Autophosphorylation at a Novel Site in the Unique Domain

Autophosphorylation is a key event in the activation of protein kinases. In this study, we demonstrate that autophosphorylation of the recombinant Src family kinase Hck leads to a 20-fold increase in its specific enzymatic activity. Hck was found to autophosphorylate readily to a stoichiometry of 1.3 mol of phosphate per mol of enzyme, indicating that the kinase autophosphorylated at more than one site. Solid phase sequencing and two-dimensional mapping of the phosphopeptide fragments derived from the autophosphorylated enzyme revealed that the kinase can undergo autophosphorylation at the following two sites: (i) Tyr-388, which is located to the consensus autophosphorylation site commonly found in the activation loop of many protein kinases, and (ii) Tyr-29, which is located in the unique domain of Hck. Hck purified from mouse bone marrow-derived macrophages could also autophosphorylate in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29. Furthermore, Hck transiently expressed in human embryonic kidney 293T cells was found to be phosphorylated at Tyr-29 and Tyr-388, proving that Hck can also undergo autophosphorylation at both sites in vivo. The recombinant enzyme carrying the mutation of Tyr-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a significantly slower rate. A 2-fold increase in the specific enzymatic activity was seen with this mutant despite the stoichiometry of autophosphorylation only approaching 0.2 mol of phosphate per mol of enzyme. This indicates that autophosphorylation of Tyr-29 contributes significantly to the activation of Hck. Regulation of the catalytic activity by phosphorylation of Tyr-29 in the unique domain may represent a new mechanism of regulation of Src family tyrosine kinases.

Nitric Oxide Prevents Tumor Necrosis Factor α–Induced Rat Hepatocyte Apoptosis by the Interruption of Mitochondrial Apoptotic Signaling Through S-Nitrosylation of Caspase-8

Mitochondrial cytochrome c release plays a critical role in apoptotic signal cascade after the activation of cell surface death receptors. We investigated the role played by nitric oxide (NO) in mitochondrial apoptotic signaling in tumor necrosis factor α (TNF-α) plus actinomycin D (TNF-α/ActD)-induced apoptosis. NO produced either by S-nitroso-N-acetyl-DL-penicillamine (SNAP) or inducible NO synthase (iNOS) prevented TNF-α/ActD-induced apoptosis in hepatocytes and also inhibited both caspase-8–like (IETDase) and caspase-3–like protease (DEVDase) activity as well as mitochondrial cytochrome c release. Recombinant human (rh) caspase-8 induced the cleavage of the cytochrome c–effluxing factor Bid and cytochrome c release from purified mitochondria in the reconstitution system with Bid+/+ cytosol, but not with Bid−/− cytosol. The addition of SNAP and the caspase-8 inhibitor Ac-IETD-fmk inhibited caspase-8–dependent Bid cleavage and cytochrome c release. The inhibitory effect of NO on caspase-8 was reversed by dithiothreitol (DTT). Furthermore, rh-caspase-8 was found to be modified by S-nitrosylation with 1.7 moles of NO bound per mole of enzyme. Treatment of hepatocytes with interleukin 1β (IL-1β) plus interferon gamma (IFN-γ), which induced iNOS expression and NO production, suppressed TNF-α/ActD-induced Bid cleavage and mitochondrial cytochrome c release. The NOS inhibitor NG-monomethyl-L-arginine (NMA) inhibited the protective effects of IL-1β and IFN-γ. The liver-specific NO donor V-PYRRO/NO also inhibited in vivo elevation of IETDase activity, Bid cleavage, and mitochondrial cytochrome c release in the livers of rats injected with TNF-α plus D-galactosamine. Our results indicate that one mechanism by which NO protects hepatocytes from TNF-α/ActD-induced apoptosis is via the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.(Hepatology 2000;32:770-778.)

Action of Metalloproteinases Mutalysin I and II on Several Components of the Hemostatic and Fibrinolytic Systems

The zinc endopeptidases mutalysin I (100 kDa) and mutalysin II (22.5 kDa) have been previously isolated from bushmaster ( Lachesis muta muta) snake venom. Hemorrhagic activity was observed with as little as 0.5 μg (2000 units/mg) and 17.8 μg (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the Aα>Bβ chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5.25 and 16.3 μmol fibrinogen/min/μmol protein for mutalysin I and II, respectively. In vitro, the enzymes act directly on fibrin and are not inhibited by serine proteinase inhibitors (SERPINS). Analysis by SDS-PAGE of fibrin hydrolysis by both enzymes showed that mutalysin II (0.22 μM) completely digested the α- and γ-γ chains and partially the β-chain (in 120 min incubation). In contrast, mutalysin I (three fold higher concentration than mutalysin II) hydrolyzed selectively the α-chain of fibrin leaving the β and γ-γ chains unaffected. Unlike with the plasminogen activator-based thrombolytic agents (e.g., streptokinase), mutalysins do not activate plasminogen. Neither enzyme had an effect on protein C activation. Mutalysin II does not inhibit platelet aggregation in human PRP induced by collagen or ADP. However, mutalysin I showed a selective inhibitory effect on collagen-induced aggregation of human PRP; it did not affect platelet aggregation with ADP as the agonist. The present investigation demonstrates that both native and EDTA-inactivated mutalysin I dose dependently blocked aggregation of human PRP elicited by 10 μg/mL of collagen with an IC 50 of 180 and 580 nM, respectively. These studies suggest that, in addition to the metalloprotease region of mutalysin I, the disintegrin-like domain also participates in the inhibitory effect. The proteolytic activity of mutalysin II against dimethylcasein and fibrin was completely abolished by α2-macroglobulin (α2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of α2-M. In contrast, the proteolytic effect of mutalysin I against the same substrates was not significantly inhibited by α2-M. Therefore, the data explain why mutalysin I contributes significantly not only to local but also to systemic bleeding associated with the observed pathological effects of the venom.

2,4-Dienoyl-CoA Reductase from Escherichia coli Is a Novel Iron–Sulfur Flavoprotein That Functions in Fatty Acid β-Oxidation

2,4-Dienoyl-CoA reductase is an enzyme that is required for the β-oxidation of unsaturated fatty acids with even-numbered double bonds. The 2,4-dienoyl-CoA reductase from Escherichia coli was studied to explore the catalytic and structural properties that distinguish this enzyme from the corresponding eukaryotic reductases. The E. coli reductase was found to contain 1 mol of flavin mononucleotide and 4 mol each of acid-labile iron and sulfur in addition to 1 mol of flavin adenine dinucleotide per mole of protein. Redox titrations revealed a requirement for 5 mol of electrons to completely reduce 1 mol of enzyme and provided evidence for the formation of a red semiquinone intermediate. The reductase caused a significant polarization of the substrate carbonyl group as indicated by an enzyme-induced red shift of 38 nm in the spectrum of 5-phenyl-2,4-pentadienoyl-CoA. However, suspected cis → trans isomerase and Δ3,Δ2-enoyl-CoA isomerase activities were not detected in this enzyme. It is concluded that the 2,4-dienoyl-CoA reductases from E. coli and eukaryotic organisms are structurally and mechanistically unrelated enzymes that catalyze the same type of reaction with similar efficiencies.

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.

Expression and purification of recombinant human indoleamine 2, 3-dioxygenase.

Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tryptophan metabolism, has been implicated in the pathogenesis of many diseases. The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a fusion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosphocellulose and nickel-agarose affinity chromatography. The yield of the fusion protein was 1.4 mg per liter of bacterial culture with an overall recovery of 56% from the crude extract. When the culture medium was supplemented with 7 microM hemin, the purified protein contained 0.8 mol of heme per mole of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein. The pI value of the recombinant enzyme was 7.09 compared with 6.9 for the native enzyme. This was as expected from the addition of the hexahistidyl tag. Similar to the native enzyme, the recombinant enzyme required methylene blue and ascorbic acid for enzyme activity and oxidized not only l-tryptophan but also d-tryptophan and 5-hydroxy-l-tryptophan. The molecular activities for these substrates and their K(m) values were similar to those of the native enzyme, indicating that the addition of the hexahistidyl tag did not significantly affect catalytic activity. The recombinant protein can therefore be used to investigate properties of the native enzyme. This will aid the development of specific inhibitors of indoleamine 2,3-dioxygenase, which may be effective in halting disease progression.

Chemical modification of tryptophan residues of d-amino acid oxidase from Rhodotorula gracilis

d-Amino acid oxidase was inactivated by N-bromosuccinimide (NBS) at 30°C and pH 8. The reaction followed pseudo-first order kinetics with second-order rate constants of 69.8 mM −1 min −1 for the apoenzyme and 0.63 mM −1 min −1 for the holoenzyme. The presence of substrates or benzoate protected the enzyme against inactivation. Difference absorption spectra at 280 nm, low consumption of NBS per mole of enzyme, the decrease in the fluorescence emission at 335 nm, integrity of the protein backbone and the absence of cysteine oxidation pointed to the modification of tryptophan residues. The statistical analysis of the residual fractional activity vs. the number of modified tryptophan residues led to the conclusion that one tryptophan residue is essential for the enzyme activity. This tryptophan residue was not involved in binding of FAD or dimerization of the enzyme.

Tyrosinase inactivation in organic solvents

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction.

Tyrosinase inactivation in organic solvents

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 × 103 mol product/((mol enzyme)(min)) in toluene and 8.7 × 103 mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 × 103 mol product/mol enzyme in toluene and 7.2 × 104 mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 325–333, 1999.

The Use of Fluorescein 5′-Isothiocyanate for Studies of Structural and Molecular Mechanisms of Soybean Lipoxygenase

Incubation of fluorescein 5′-isothiocyanate (FITC) with soybean lipoxygenase produces the coupling of 1 mol of fluorophore to 1 mol of enzyme. Derivatized lipoxygenase lost 40% activity through a mixed-type inhibitory mechanism. The quenching by IK of the fluorescence of FITC-labeled lipoxygenase shows that the fluorophore is located near the surface of the protein. The partial impediment of the FITC labeling when the substrate is present in the medium, together with data of the tryptic digestion, suggests that FITC is attached via the access channel of substrate to the catalytic site. Labeling does not induce appreciable modification of the enzyme specificity, suggesting that the position of substrate in the active site is not modified by the labeling. The results obtained strongly suggest that FITC labels soybean lipoxygenase specifically at a lysyl residue which contributes to fixation of the carboxylic end of the substrate to the active center. The experimental data obtained from the quenching of FITC fluorescence by NDGA reveal that this molecule interacts with the protein near the catalytic site.

Phospholipase A(2) digestion of cardiolipin bound to bovine cytochrome c oxidase alters both activity and quaternary structure.

Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme.

Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U x mg protein-1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 microM. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U x mg protein-1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 microM. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 microM. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.

Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein.

Flavonol 2,4-dioxygenase, which catalyzes the cleavage of quercetin to carbon monoxide and 2-protocatechuoyl-phloroglucinol carboxylic acid, was purified from culture filtrate of Aspergillus niger DSM 821 grown on rutin. It is a glycoprotein (46-54% carbohydrate) with N-linked oligo-mannose type glycan chains. The enzyme was resolved in SDS polyacrylamide gels in a diffuse protein band that corresponded to a molecular mass of 130-170 kDa. When purified flavonol 2,4-dioxygenase was heated, it dissociated into three peptides with apparent molecular masses of 63-67 kDa (L), 53-57 kDa (M), and 31-35 kDa (S), which occurred in a molar ratio of 1:1:1, suggesting a LMS structure. Crosslinking led to a 90-97 kDa species, concomitant with the decrease of staining intensity of the 63-67 kDa (L) and the 31-35 kDa (S) peptides. Analysis by matrix-assisted laser desorption/ionization-time of flight-MS showed peaks at m/z approximately 69 600, m/z approximately 51 700, and m/z approximately 26 500 which are presumed to represent the three peptides of flavonol 2,4-dioxygenase, and a broad peak at m/z approximately 96 300, which might correspond to the LS heterodimer as formed in the crosslinking reaction. Based on the estimated molecular mass of 148 kDa, 1 mol of enzyme contained 1.0-1.6 mol of copper. Ethylxanthate, which specifically reduces CuII to CuI ethylxanthate, is a potent inhibitor of flavonol 2,4-dioxygenase. Metal chelating agents (such as diethyldithiocarbamate, diphenylthiocarbazone) strongly inhibited the enzymatic activity, but inactivation was not accompanied by loss of copper. The EPR spectrum of flavonol 2,4-dioxygenase (as isolated) showed the characteristic parameters of a nonblue type 2 CuII protein. The Cu2+ is assumed to interact with four nitrogen ligands, and the CuII complex has a (distorted) square planar geometry.

DNA-dependent RNA polymerase II from Candida species is a multiple zinc-containing metalloenzyme.

We have purified DNA-dependent RNA polymerase II from Candida albicans, a human pathogenic yeast. The enzyme consists of 9 polypeptides that are unique to C. albicans, their mobility on SDS-PAGE being different from the mobility of the corresponding subunits of RNA polymerase II from Saccharomyces cerevisiae or C. utilis. In the present study we also demonstrate that RNA pol II from C. albican and C. utilis are metalloproteins containing approximately 5 mol of zinc per mole of enzyme. Although prolonged dialysis in 10 or 20 mM EDTA failed to remove Zn(II) from the C. albicans enzyme, in the C. utilis enzyme 3 Zn(II) ions could be removed and then reconstituted in the presence of excess Zn(II). o-Phenanthroline (5 mM) removed Zn(II) from C. albicans enzyme irreversibly in a time-dependent fashion with concomitant loss of enzyme activity. Circular dichroism studies revealed structural changes on removal of zinc, thus suggesting a role for Zn in maintenance of structural stability. Further, we demonstrate that the largest subunit of the C. utilis enzyme and the 3 large subunits of the C. albicans enzyme can bind radioactive zinc.