Affinity labeling of rabbit muscle pyruvate kinase with dialdehyde-ADP.

Periodate-oxidized ADP (dialdehyde-ADP) inactivates rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and combines irreversibly to the enzyme. This inactivation is first-order with respect to dialdehyde-ADP and follows saturation kinetics, indicating that the enzyme first forms a reversible complex with the inactivator. Low Mg2+ concentrations stimulate the rate of inactivation, while higher concentrations have a protective effect. ADP and ATP, especially in the presence of Mg2+, protect very strongly against inactivation, while phosphoenolpyruvate and pyruvate are less effective. Dialdehyde-ADP is not a substrate, but acts as competitive inhibitor of ADP, with a KI of 4.5 mM. The analog has somewhat lower affinity to the enzyme than Mg-ADP, which has a Kd of 1.2 mM. Based on kinetic data, it is shown that one molecule of reagent must combine per enzyme active site in order to inactivate the enzyme. Incorporation of [14-C]dialdehyde-ADP to the enzyme and treatment of the data by the Tsou plot shows that 6-7 residues per subunit react with the modifier, two of them being essential for activity. From the evidence presented it is concluded: (1) dialdehyde-ADP behaves as an affinity label of rabbit muscle pyruvate kinase; (2) the inactivator binds probably to lysine residues at or near the active site, forming morpholine-like structures, and (3) the enzyme possesses two modifiable groups essential for activity, the reaction of one of them being sufficient to cause total loss in activity.

Cloning, sequencing and expression of the cDNA of endoxylanase B from Penicillium purpurogenum.

The cDNA for xylanase B from Penicillium purpurogenum was cloned and sequenced. This DNA encodes a protein of 208 amino acids which is expected to yield a protein of 183 residues upon processing of the N terminus. The sequence of the predicted protein is very similar to that of 40 other xylanase domains which belong to family G of cellulases/xylanases (73-21% identity).

Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain.

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.

Electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum and chromosomal location of several xylanolytic genes.

The electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum has been resolved. Using contour-clamped homogeneous electric field gel electrophoresis, five chromosomal bands were separated, with estimated sizes of 7.1, 5.2, 3.7, 2.9 and 2.3 Mbp, giving a total genome size of 21.2 Mbp. To our knowledge, this is the smallest Penicillium genome determined so far. By Southern blots and using homologous probes, the chromosomal location of five xylanolytic genes from P. purpurogenum was determined: axeI (acetyl xylan esterase I), xynB (endoxylanase B) and abf1 (arabinofuranosidase 1) in chromosome I, xynA (endoxylanase A) in chromosome II, and axeII (acetyl xylan esterase II) in chromosome III. This is the first study where the location of xylanase genes in a Penicillium genome has been established.

An alpha-L-arabinofuranosidase from Penicillium purpurogenum: production, purification and properties.

Penicillium purpurogenum secretes arabinofuranosidase to the growth medium. Highest levels of enzyme (1.0 U ml(-1)) are obtained when L-arabitol is used as carbon source, while 0.85 and 0.7 U ml(-1) are produced with sugar beet pulp and oat spelts xylan, respectively. By means of a zymogram, three bands with arabinofuranosidase activity have been detected in the supernatant of a culture grown in oat spelts xylan. One of the enzymes was purified to homogeneity from this supernatant using gel filtration (BioGel P-100), cation exchange chromatography (CM-Sephadex C-50), hydrophobic interaction chromatography (phenyl agarose) and a second BioGel P-100 column. The enzyme is a monomer of 58 kDa with a pI of 6.5. Optimum pH is 4.0 and optimal temperature 50 degrees C. The arabinofuranosidase is highly specific for alpha-L-arabinofuranosides and liberates arabinose from arabinoxylan. The enzyme shows hyperbolic kinetics towards p-nitrophenyl-alpha-L-arabinofuranoside with a K(M) of 1.23 mM. A 36-residue N-terminal sequence is over 70% identical to that of fungal arabinofuranosidases belonging to family 54 of the glycosyl hydrolases. Based on the sequence similarity and other biochemical properties it is proposed that the purified enzyme from P. purpurogenum belongs to family 54.

Penicillium purpurogenum produces several xylanases: purification and properties of two of the enzymes.

The fungus Penicillium purpurogenum produces several extracellular xylanases. The two major forms (xylanases A and B) have been purified and characterized. After ammonium sulfate precipitation and chromatography in Bio-Gel P 100, xylanase A was further purified by means of DEAE-cellulose, hydroxylapatite and CM-Sephadex, and xylanase B by DEAE-cellulose and CM-Sephadex. Both xylanases showed apparent homogeneity in SDS-polyacrylamide gel electrophoresis. Xylanase A (33 kDa) has an isoelectric point of 8.6, while xylanase B (23 kDa) is isoelectric at pH 5.9. Antisera against both enzymes do not cross-react. The amino terminal sequences of xylanases A and B show no homology. The results obtained suggest that the enzymes are produced by separate genes and they may perform different functions in xylan degradation.

An overview on chemical modification of enzymes. The use of group-specific reagents.

Chemical modification is an important approach to the study of enzyme active sites. This article presents an overview of the methods used. The basic concepts, applications and limitations of chemical modification are outlined. The use of reagents specific for different amino acid side chains is also discussed.

Culture conditions for enhanced cellulase production by a native strain of Penicillium purpurogenum.

A cellulolytic wild-type strain of Penicillium purpurogenum was isolated from a soil sample in southern Chile. It grew best at 28°C from an inoculum of 4×10(7) spores/100 ml medium. Highest endoglucanase activity was with Sigmacell as carbon source and corn steep liquor as nitrogen source. Wheat bran enhanced the production of endoglucanase and ß-glucosidase. The enzymes in the crude supernatants were stable up to 50°C and between pH 4.4 and 5.6 for 48 h.

Beta-glucosidase from Penicillium purpurogenum: purification and properties.

beta-Glucosidase was purified from the culture supernatant of Penicillium purpurogenum. The purified enzyme was homogeneous on both nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme is a monomeric glycoprotein with M(r) of 90,000 as determined by gel filtration on Bio-Gel P-300 and SDS-polyacrylamide gels. Two enzyme forms were resolved by chromatofocusing and isoelectric focusing, and the pI values obtained with both methods were 4.2 (major form) and 6.0. The major form was characterised further. Enzyme activity was optimal at pH 3.5 and at 60 degrees C. The enzyme was stable in the pH range 2.5-9.5 for 24 h at 4 degrees C. Kinetic analysis gave Kms of 0.8 mM for cellobiose and 85 microM for p-nitrophenyl-beta-D-glucopyranoside. The enzyme hydrolyses a wide range of substrates including aryl-beta-glucosides, cellobiose, and amygdalin. Glucose inhibits competitively and glucono-delta-lactone is a mixed inhibitor of the enzyme.

Two malic enzymes in Pseudomonas aeruginosa.

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) --> pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO(2)), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn(2+) being more effective than Mg(2+). The NADP-dependent enzyme is activated by K(+) and low concentrations of NH(4) (+). Both reactions are reversible, as shown by incubation with pyruvate, CO(2), NADH, or NADPH and Mn(2+). The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).

Purification and characterization of avian liver mevalonate-5-pyrophosphate decarboxylase.

Mevalonate-5-pyrophosphate decarboxylase [ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33] has been purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of molecular weight 85400 +/- 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for adenosine 5'-triphosphate (ATP) and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0 to 6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column chromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM have been obtained for mevalonate-5-pyrophosphate and ATP, respectively.

Evidence of essential arginyl residues in chicken liver mevalonate-5-pyrophosphate decarboxylase.

Chicken liver mevalonate-5-pyrophosphate decarboxylase (ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33.) is inactivated by phenylglyoxal in triethanolamine buffer at pH 8.15. The reaction follows pseudo-first-order kinetics with a second-order rate constant of 108 M-1 min-1. Appropriate treatment of the kinetic data for the inactivation reaction indicates that the reaction of a single phenylglyoxal molecule per active unit of the enzyme is enough to completely inactivate the protein. The partially inactivated enzyme shows unaltered Km but decreased V as compared to native mevalonate-5-pyrophosphate decarboxylase. The dissociation constants for the enzyme-substrate complexes were estimated from inactivation reactions at different concentrations of substrates. From the data it is concluded that the modified amino acid is important for the binding of both substrates.

Photoaffinity labeling of pyruvate kinase from rabbit muscle.

Several studies have been performed on the structure of muscle pyruvate kinase. X-ray diffraction has provided a three-dimensional picture of the active site, and chemical modification studies have revealed essential amino acid residues for substrate binding or catalysis. We have shown that 8-azido-ADP (N3 ADP) behaves as a photoaffinity label for the enzyme. This reagent upon irradiation produces inactivation of the enzyme, and the activity loss is protected by nucleotides. The partially modified enzyme shows the same Km for ADP as the native one suggesting an "all or none" inactivation effect. The incorporation of 1 mole of 14C-N3 ADP per subunit correlates with complete inactivation. A radioactive peptide was isolated from the enzyme labeled with 14C-N3 ADP. The partial sequence of this peptide showed that it corresponds to the same peptide isolated from rabbit muscle pyruvate kinase labeled with dialdehyde-ADP and with trinitrobenzenesulfonate. This peptide is identical to a region in the cat and chicken muscle enzymes, and also a high degree of homology is found in a region of the rat liver and yeast enzymes. These studies show that N3 ADP binds to the same site as dialdehyde-ADP in rabbit muscle pyruvate kinase, and this site seems to be the nucleotide binding site.

Purification and characterization of two acetyl xylan esterases from Penicillium purpurogenum.

Penicillium purpurogenum produces several enzymes active in xylan hydrolysis, of there, the acetyl xylan esterase (AXE) activity secreted by the fungus has now been studied. The amount of activity obtained in the culture is related to the degree of acetylation of the carbon source used, the best being chemically acetylated xylan. AXE was concentrated from culture supernatants by ultrafiltration and (NH4)2SO4 precipitation and fractionated by gel filtration in Bio-Gel P-300. Two peaks of activity (AXE I and AXE II) were obtained. These two enzymes were further purified separately to homogeneity by chromatography in CM-Sephadex C-50 and chromatofocusing. AXE I (M(r) 48,000) has a pl of 7.5, while AXE II (M(r) 23,000) has a pl of 7.8. Optimal enzyme activity was at pH 5.3 and 50 degrees C for AXE I and pH 6.0 and 60 degrees C for AXE II. Both enzymes are active towards several acetylated substrates. Antisera against the two enzymes do not cross-react, and the N-terminal sequences of AXE I and II do not show similarities. These results suggest that AXE I and AXE II are the products of different genes.

Structure analysis of the endoxylanase A gene from penicillium purpurogenum.

Penicillium purpurogenum produces several endoxylanases, two of which (XynA and XynB) have been purified and characterized. XynB has been sequenced, and it belongs to glycosyl hydrolase family 11. In this publication we report the structure of the xynA gene. The amino terminal sequence of the protein was determined and this allowed the design of oligonucleotides for use in polymerase chain reactions. Different polymerase chain reaction strategies were used to amplify and sequence the entire cDNA and the gene. The gene has an open reading frame of 1450 base pairs, including 8 introns with an average length of 56 base pairs each. Only one copy of this gene is present in the P. purpurogenum genome as shown by Southern blot. The gene encodes a protein of 329 residues (including the signal peptide), and the calculated molecular mass of the mature protein is 31,668 Da. Immunodetection assays of the expressed gene positively identified it as xynA, and sequence alignments indicate a high degree of similarity with family 10 endoxylanases. It is concluded that P. purpurogenum produces endoxylanases of family 10 and 11. The complementary action of endoxylanases of both families may be important for an efficient degradation of xylan by the fungus.

Pyruvate kinase: studies on affinity labeling and active-site structure using the rabbit muscle enzyme.

Important advances have been made in recent years in the study of the structure of pyruvate kinase: the amino acid sequence of the enzymes from chicken muscle and yeast have been established and the three-dimensional structure of the cat muscle enzyme has been determined at 0.26 nm resolution. Work in our laboratory has shown that dialdehyde-ADP (oADP) can be used as an affinity label of rabbit muscle pyruvate kinase: if the enzyme is incubated with cold oADP in the presence of high ADP concentrations, dialyzed and then incubated with 14C-oADP, the enzyme inactivates and one mole of radioactive oADP incorporates per mole of enzyme subunit. A labeled peptide with a molecular weight of about 5900 has been purified from a tryptic digest of the modified enzyme. The first 26 residues of the peptide have been sequenced and this sequence is identical to a region in the chicken muscle enzyme and a peptide isolated from the bovine muscle enzyme specifically labeled with trinitrobenzenesulfonate. High homology is also found with a region of the yeast enzyme. All this suggests that the isolated peptide is part of the active site; the modified amino acid, probably a lysine, seems to be located in one of the alfa helices of domain A of the enzyme, according to the x-ray data.

Yeast pyruvate kinase: essential lysine residues in the active site.

1. Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by trinitrobenzenesulfonate and by P1, P2-bis (5' pyridoxal) diphosphate. 2. Labeled peptides were isolated and their amino acid composition was determined. 3. The results suggest that yeast pyruvate kinase has an essential lysine residue, and that this residue is in a location equivalent to an essential lysine described in the muscle enzyme. 4. Protection experiments indicate that this lysine is located at the nucleotide binding site.

Pig liver phosphomevalone kinase. 1. Purification and properties.

Pig liver phosphomevalonate kinase (EC 2.7.4.2) has been purified to homogeneity as shown by polyacrylamide gel electrophoresis. The molecular weight estimates range from 21,000 to 22,500. Each molecule is composed of one polypeptide chain. The presence of SH-containing reagents is essential for the preservation of enzymes activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM have been obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, one cysteine residue per molecule of enzyme, and the absence of methionine. The results obtained suggest that the enzyme plays no regulatory function in cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.

Determination of a protein structure by iodination: the structure of iodinated acetylxylan esterase.

Enzymatic and non-enzymatic iodination of the amino acid tyrosine is a well known phenomenon. The iodination technique has been widely used for labeling proteins. Using high-resolution X-ray crystallographic techniques, the chemical and three-dimensional structures of iodotyrosines formed by non-enzymatic incorporation of I atoms into tyrosine residues of a crystalline protein are described. Acetylxylan esterase (AXE II; 207 amino-acid residues) from Penicillium purpurogenum has substrate specificities towards acetate esters of D-xylopyranose residues in xylan and belongs to a new class of alpha/beta hydrolases. The crystals of the enzyme are highly ordered, tightly packed and diffract to better than sub-angström resolution at 85 K. The iodination technique has been utilized to prepare an isomorphous derivative of the AXE II crystal. The structure of the enzyme determined at 1.10 A resolution exclusively by normal and anomalous scattering from I atoms, along with the structure of the iodinated complex at 1.80 A resolution, demonstrate the formation of covalent bonds between I atoms and C atoms at ortho positions to the hydroxyl groups of two tyrosyl moieties, yielding iodotyrosines.