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The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid. The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa. Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species. Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs. MDHs and LDHs resemble each other in catalytic mechanism and several other respects. However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs. The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric. A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E. coli plasmid vector. BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E. coli strain carrying the clone. MDH purified from BI and that purified from the E. coli strain carrying the MDH gene clone appear to be identical proteins by several criteria. A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters. Whereas the MDH is a stable tetramer in the pH range 5–7, it appears to be converted into a stable dimer at pH 3.5. This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH.

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2′- and 3′-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.

The stability of wild-type Escherichia coli malate dehydrogenase was compared with a mutant form of the enzyme with the amino acid residue at position 102 changed from arginine to glutamine. The mutation occurs on the underside of a mobile loop which closes over the active-site cleft on formation of the enzyme/cofactor/substrate ternary complex. The mutant enzyme is kinetically compromised while the wild-type enzyme is highly specific for oxaloacetate. The mutant enzyme was shown to be more resistant to irreversible thermal denaturation by thermal inactivation experiments and high-sensitivity differential scanning calorimetry than the wild-type enzyme. In contrast, resistance of both enzymes to reversible unfolding in guanidinium chloride was similar. Circular dichroic spectropolarimetry shows the secondary structures of the enzymes are similar but there is a demonstrable difference in tertiary structure. From the position of the mutation, it is conjectured that the substitution on a mobile surface loop results in partial closure of the loop and greater resistance to thermal inactivation of the mutant enzyme. However, molecular modelling combined with circular dichroic spectropolarimetry indicate that the mutation may have a more widespread effect on the structure than simply partial closure of the mobile surface loop as the environment of distant tyrosine residues is altered. Resistance of the wild-type enzyme to thermal inactivation can be increased by cofactor addition, which may have the effect of partial closure of the mobile surface loop, but has little effect on the mutant enzyme.

Unlike the European leech Hirudo medicinalis, the Asian jawed leech Hirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions of Hirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as with Hirudo leech, various isoforms of hirudin also exist in Hirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.

X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme. The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195. Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S.,Beppu, T. (1991) J. Biol. Chem. 266, 14294-14299].

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the formation of a salt bridge with a carboxylate group of oxaloacetate.

Evidence is presented to demonstrate that the Zn2+ metallo-enzyme glycerol dehydrogenase from the thermophile Bacillus stearothermophilus has one cysteine residue per subunit which is only available for reaction with thiol reagents in the metal-depleted form of the enzyme. Modification of the metal-depleted enzyme by methyl methanethiosulphonate prevents the reactivation of the enzyme by Zn2+ ions and induces dissociation of the oligomer into subunits. The rate of reaction of the cysteine residue with the thiol reagent DTNB is limited by a factor other than reagent concentration and it is proposed that the reagent only reacts with the cysteine residue in dissociated monomers. The enzyme has been labelled at the single cysteine residue by radioactive iodo[2-3H]acetic acid. Two radiolabelled peptides have been isolated and sequenced; one peptide is a component of the other. Spectroscopic evidence suggests that the cysteine residue is not involved in ligation of the essential metal ion. Chemical modification studies using the reagent diethylpyrocarbonate have suggested that two histidines are involved in the ligation of the metal.

For an enzyme to be employed as a reagent in any field, be it clinical chemistry or organic synthesis, it must first be purified to a degree that removes any other enzyme capable of catalysing undesirable side-reactions. This may or may not mean purification to homogeneity. For the enzyme to be commercially viable, purification must yield tens or hundreds of grams of protein. On this scale, the availability of sufficient starting material may pose a problem, particularly for animal tissues. For this and other reasons, bacteria may provide the most valuable source of enzymes. Bacteria are readily produced in enormous quantities and, by virtue of their diverse metabolism, contain many enzymes not present in other organisms. Furthermore, the enzymes from thermophilic species often show a desirable increase in stability under the arduous conditions which may be encountered in a reactor. The preparation of enzymes on this scale from bacteria, or any other source, can usually be accomplished by the application of the normal range of techniques available to the protein chemist. Thus precipitation methods, gel filtration, ion exchange and affinity chromatography are all valuable. A major difference between this and normal laboratory-scale scale purification is cost; large-scale enzyme purification requires a considerable investment in equipment, materials and manpower. This cost must be viewed in terms of the value of the product.

This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope. Binding of two hexapeptides (283IVPPDE288 and 287DEELPG292) to the antibodies was dependent on Pro285, and Pro286, since their replacement by almost any other amino acid resulted in reduced binding. The other residues were less important for binding the antibodies, as binding was relatively unaffected by amino acid substitutions. Three site-directed mutant enzymes, P285T (proline-285-->threonine etc.), P286Q and E288A, were expressed in Escherichia coli. The purified enzymes had subunit M(r) values of 35,000. The pI values of P285T, P286Q and the wild-type enzymes were 8.6, and that for the mutant E288A was 9.2. The kcat. and Km values for the mutants P286Q and E288A with L-asparagine and L-glutamine were comparable with those of the wild-type enzyme. The Km values for the mutant P285T with both substrates was similar to that of the wild-type enzyme, whereas the kcat. was reduced by 2-fold with L-asparagine and by 4-fold with L-glutamine. The change proline-->threonine reduced the antigenicity of the enzyme by 8-fold, as shown in sandwich e.l.i.s.a.s. using monoclonal antibodies raised against the wild-type enzyme.

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

The Gin residue at amino acid position 102 ofBacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured byk cat/K m) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

A novel protein able to bind with high affinity to the Fc fragment of IgG from a variety of animals has been produced by a gene synthesis approach. The IgG binding is accomplished by the presence of a single or two consecutive domains based upon domain B from protein A of Staphylococcus aureus. The IgG-binding moiety is fused to a peptide containing 21, 53 or 81 amino acids derived from the N-terminus of bovine DNase I. The latter is present to guide the expression of the protein in Escherichia coli into an inclusion body. This facilitates the high expression and recovery of the IgG-binding domains. The binding activity of this fusion protein is very close to that of the native protein A. Site-directed mutagenesis of the fusion protein and subsequent identification of changed binding interactions is reported.

The human LDH-A and LDH-B cDNAs, containing the coding regions for the l -lactate dehydrogenase A4 (M) and B4 (H) polypeptides respectively have been cloned into Escherichia coli to place the cDNAs under the control of hybrid E. coli/Bacillus stearothermophilus transcriptional and translational signals. Human A4- and B4-isoenzymes are produced in E. coli cells harbouring the expression plasmids pHLDHA22 and pHLDHB10 at levels of 6.5 and 1.5% of the soluble protein of the cell, respectively. The tac promoter of these vectors was not induced by isopropyl β- d -thiogalactopyranoside. The A4 and B4 human isoenzymes synthesized in E. coli were purified to homogeneity and show the same properties as isoenzymes isolated from human tissue. The amino acid sequences of 12 N-terminal residues of the human isoenzymes synthesized in E. coli were determined to be identical to those deduced from the DNA sequence of the cloned cDNAs except that the N-terminal methionine was absent from both. However, in contrast to LDH made in human cells, acetylation of the N-terminal alanine does not take place in E. coli cells.

Evidence is presented to demonstrate that the Zn2+ -depleted, inactive form of the glycerol dehydrogenase from Bacillus stearothermophilus exists in one of two possible conformations in equilibrium, the position of which is temperature sensitive. The conformation of the metal-depleted enzyme favoured by higher temperatures (20-40 degrees C) is able to bind Zn2+ and regain catalytic activity, whereas that favoured at lower temperatures (0-10 degrees C) is unable to bind metal ions and is thus inactive. This equilibrium is also pH dependent with a pK of 6.6. At pH 6.0, the equilibrium lies in favour of the form of the enzyme able to bind metal ions and exhibit activity.

Basic c -type cytochromes homologous with plant and animal mitochondrial cytochrome c have been isolated and purified from Rhodymenia palmata and Porphyra umbilicalis . The N -terminal regions have been analysed using a Beckman 890C automatic sequencer. When compared to animal cytochrome c , the Rhodymenia cytochrome c has an unblocked N -terminal tail of 10 amino acids, whereas Porphyra has an unblocked N -terminal tail of only a single amino acid.

Homogeneous β-lactamase (β-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the β-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.

The complete amino acid sequence of Sambucus nigra L. plastocyanin has been determined. The protein consists of a single polypeptide chain of 99 amino acids and one atom of copper, giving a molecular weight of 10517. The sequence shows considerable similarity to that of Chlorella fusca plastocyanin.

3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were purified to homogeneity on a large scale involving only two sequential affinity-chromatography steps on two triazine dye-Sepharose matrices. Recoveries of both enzymes were in excess of 60%. Malate dehydrogenase could also be purified by a combination of triazine dye affinity chromatography and gel filtration on Ultrogel AcA-44, but this offered no significant advantage over the purely affinity procedure.

The amino acid sequence of plastocyanin from broad bean was determined. It consists of a single polypeptide chain of 99 residues. The sequence was determined by using a Beckman 890C sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic cleavage of purified cyanogen bromide fragments. Some parts of the sequence depend on the results of Edman degradation of peptides for which amino acid analyses were not obtained. The evidence for one overlap is not strong.

The amino acid sequences of the plastocyanins from Mercurialis perennis and Capsella bursa-pastoris have been determined. The amides at positions 64 and 68 in the Mercurialis sequence were positioned by ‘homology’ Both proteins are single polypeptide chains of 99 residues and are closely related to other higher plant plastocyanins.

A new and novel method for determination of serum acetaminophen is described. The assay, which can be completed in less than 5 min, is based on the enzymatic hydrolysis of acetaminophen, with subsequent colorimetric detection of the aminophenol so produced. Various possible means of aminophenol estimation are described; the final reaction conditions have been optimized for maximum sensitivity and assay speed. This assay compares favorably with other available procedures; it requires only small sample volumes; it is rapid, simple, and highly specific for the parent drug; and it requires neither great technical ability nor expensive instrumentation.

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.

Aryl acylamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon. The enzyme was purified to homogeneity by a combination of ion-exchange, hydrophobic and gel-permeation chromatography. A relative molecular mass of about 52 500 was estimated by gel filtration. The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme was maximally active at a pH of 8.6 and at a temperature of 45 degrees C. The enzyme shows Michaelis-Menten kinetics; Km values for nitroacetanilide (69 microM) and hydroxyacetanilide (6.1 microM) were low, indicating that the enzyme has a very high affinity for both substrates.

Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose. This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein. A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.

Three cysteine-rich proteins of molecular weight 4000 to 7000, containing 4 to 7 gram atoms of cadmium, zinc, and copper per mole were isolated from Pseudomonas putida growing in 3 mM cadmium. The three proteins were induced during different phases of growth, and the smallest (molecular weight 3600; 3 gram atoms of cadmium) was released into the medium when the cells lysed. The results of amino acid analyses and of ultraviolet, circular dichroism, electron paramagnetic resonance, and cadmium-113 nuclear magnetic resonance spectroscopy suggest a novel cadmium(II)-zinc(II)-copper(I) cluster structure for the major protein.