Formaldehyde dismutase, a novel NAD-binding oxidoreductase from Pseudomonas putida F61

A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F61. The enzyme catalyzes the dismutation of aldehydes and alcohol:aldehyde oxidoreduction in the absence of an exogenous electron acceptor. The enzyme is composed of four identical subunits with a Mr of 44 000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol. The ratio of NAD+ and NADH in a crystalline preparation of the enzyme was about 7:3. The enzyme-bound coenzyme was completely reduced and oxidized on the addition of a large amount of an alcohol and an aldehyde respectively. Both the oxidized and reduced enzymes catalyzed the dismutation reaction to the same extent. Steady-state kinetics of the enzyme were investigated using an oxidoreduction reaction between an alcohol and p-nitroso-N, N-dimethylaniline. The enzyme obeys a ping-pong mechanism and is competitively inhibited by an alcoholic substrate analogue, pyrazole, but not coenzyme analogues, such as AMP, N-methylnicotinamide. These results indicate that NAD(H) binds firmly (but not covalently) at each active site. The enzyme-bound NAD(H) was reduced and oxidized only by the added second substrates, alcohol and aldehyde respectively, and not by exogenous electron acceptors [including NAD(H)].     

The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation.

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.     

Preparation of an alcohol-dehydrogenase--NAD(H)--sepharose complex showing no requirement of soluble coenzyme for its activity.

1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3. A molar ratio of 1:1 between the amount of bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.     

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans

Gox2253 from Gluconobacter oxydans belongs to the short‐chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short‐chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2′‐hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253‐R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate‐binding pockets. Proteins 2014; 82:2925–2935. © 2014 Wiley Periodicals, Inc.     

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

The reaction of NADP⁺ with periodate yields a coenzyme analog that can be bound to the NADP⁺ binding site of 6-phosphogluconate dehydrogenase from Candida utilis. This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.     

Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase.

Mitochondrial NADH dehydrogenase has been purified to homogeneity by resolution of Complex I from beef heart mitochondria with the chaotrope NaClO₄ and precipitation of the enzyme with ammonium sulfate. The enzyme is water-soluble, has a molecular weight of 69,000 ± 1000 as determined by gel filtration on Sephadex G-100 and agarose 1.5 M. It is an iron-sulfur flavoprotein, with the ratio of flavin (FMN) to nonheme iron to labile sulfide being 1:5–6:5–6. The FMN content suggests a minimum molecular weight of 74,000 ± 3000 for the enzyme. NADH dehydrogenase is composed of three subunits with apparent M_r values, as determined by acrylamide gel electrophoresis as well as by gel filtration on agarose 5 M both in the presence of sodium dodecyl sulfate, of about 51,000, 24,000, and 9–10,000. Coomassie blue stain intensities of the subunits on acrylamide gels suggest that they are present in NADH dehydrogenase in equimolar amounts. However, summation of the apparent M_r values of the dodecyl sulfate-treated subunits appears to overestimate the molecular weight of the native enzyme. The amino acid compositions of NADH dehydrogenase and of each of the isolated and purified subunits have been determined. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH by quinones, ferric compounds, and NAD (3-acetylpyridine adenine dinucleotide was used). All the activities of NADH dehydrogenase are greatly stimulated by addition of guanidine (up to 150 mm), alkylguanidines, arginine, and arginine methyl ester to the assay medium. Phosphoarginine had no effect. These results pointed to the importance of the positively charged guanido group, which appears to interact with and neutralize the negative charges on NAD(P)H and thereby allow for better enzyme-substrate interaction. In the absence of guanidine, NADPH is essentially unoxidized by the enzyme at pH values above 6.0. However, both NADPH dehydrogenase and NADPH → NAD transhydrogenase activities increase dramatically as the assay pH is lowered below pH = 6. Since the pK of the 2′-phosphate of NADPH is 6.1, it appears that the above pH effect is related to protonation of the 2′-phosphate, thus rendering NADPH a closer electronic analog of NADH, which is the primary substrate of the enzyme.     

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding

Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60°C and specific activity of about 2,600 U/mg with optimal temperature of 90°C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme''s activity.     

Characterization of rat cornea aldehyde dehydrogenase

Aldehyde dehydrogenase has been purified from rat cornea in a single step. The enzyme is a class 3 aldehyde dehydrogenase. Cornea aldehyde dehydrogenase is a 100-kDa dimer composed of 51-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes as well as medium chain length (4-9 carbons) aliphatic aldehydes. The substrate and coenzyme specificity, immunochemical properties, effect of disulfiram, pH profile, and isoelectric point of cornea aldehyde dehydrogenase are identical to those of tumor-associated aldehyde dehydrogenase, the prototype class 3 enzyme. The substrate and coenzyme preferences are consistent with a role for cornea aldehyde dehydrogenase in the oxidation of a variety of aldehydes generated by lipid metabolism, including lipid peroxidation.     

The crystal structure and stereospecificity of levodione reductase from Corynebacterium aquaticum M-13

The (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase (LVR) of the soil isolate bacterium Corynebacterium aquaticum M-13 is a NAD(H)-linked enzyme that catalyzes reversible oxidoreduction between (4R)-hydroxy-(6R)-2,2,6-trimethylcyclohexanone (actinol) and levodione. Here the crystal structure of a ternary complex of LVR with NADH and its inhibitor 2-methyl-2,4-pentanediol has been determined by molecular replacement and refined at 1.6-A resolution with a crystallographic R factor of 0.199. The overall structure is similar to those of other short-chain alcohol dehydrogenase/reductase enzymes. The positions of NADH and 2-methyl-2,4-pentanediol indicate the binding site of the substrate and identify residues that are likely to be important in the catalytic reaction. Modeling of the substrate binding in the active site suggests that the specificity of LVR is determined by electrostatic interactions between the negatively charged surface of Glu-103 of LVR and the positively charged surface on the re side of levodione. Mutant LVR enzymes in which Glu-103 is substituted with alanine (E103A), glutamine (E103Q), asparagines (E103N), or aspartic acid (E103D) show a 2-6-fold increase in Km values as compared with wild-type LVR and a much lower enantiomeric excess of the reaction products (60%) than the wild-type enzyme (95%). Together, these data indicate that Glu-103 has an important role in determining the stereospecificity of LVR.     

Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.     

L-erythrulose production by oxidative fermentation is catalyzed by PQQ-containing membrane-bound dehydrogenase

Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from mesoerythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized mesoerythritol to L-erythrulose irreversibly with almost 100% of recovery at 37 degrees C. L-Erythrulose was also produced efficiently by the resting cells at 37 degrees C with 85% recovery. The enzyme responsible for mesoerythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L-erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound mesoerythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between mesoerythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0-10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production.     

Studies of coenzyme binding to rabbit muscle glyceraldehyde-3-phoshate dehydrogenase.

The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+. Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.     

Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes

Gluconate 5‐dehydrogenase (Ga5DH) is an NADP(H)‐dependent enzyme that catalyzes a reversible oxidoreduction reaction between D ‐gluconate and 5‐keto‐D ‐gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. Despite the considerable amount of physiological and biochemical knowledge of Ga5DH, there is little physical or structural information available for this enzyme. To this end, we herein report the crystal structures of Ga5DH from pathogenic Streptococcus suis serotype 2 in both substrate‐free and liganded (NADP⁺/D ‐gluconate/metal ion) quaternary complex forms at 2.0 Å resolution. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer that comprised four identical ～29 kDa subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in the binding and orientation of substrate. The quaternary complex structure provides the first clear crystallographic evidence for the role of a catalytically important serine residue and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Detailed analysis of the crystal structures reveals important contributions of Ca²⁺ ions to active site formation and of specific residues at the C‐termini of subunits to tetramer assembly. Because Ga5DH is a potential target for therapy, our findings provide insight not only of catalytic mechanism, but also suggest a target of structure‐based drug design.     

Octyl glucoside induced formation of the molten globule-like state of glutamate dehydrogenase.

The interaction between n-octyl-beta-D-glucopyranoside (octyl glucoside) and bovine liver glutamate dehydrogenase (GDH) was studied using techniques including equilibrium dialysis, UV-spectrophotometry, circular dichroism (CD), fluorescence energy transfer and extrinsic spectrofluorometry in 50 mM sodium phosphate buffer solution (pH 7.6). The equilibrium dialysis experiment showed a higher binding of octyl glucoside to GDH that induces up to 80% enzyme inhibition in 20 mM octyl glucoside solution. The CD study indicated that GDH retains its secondary structure in the presence of octyl glucoside, but loses a degree of its tertiary structure by acquiring a more extended tertiary structure. Measurement of the binding of a hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to GDH revealed that the binding of ANS to GDH is increased in the presence of octyl glucoside, a finding that may be interpreted in terms of the increment of surface hydrophobic patch(es) of GDH because of its binding to octyl glucoside. Fluorescence energy transfer studies also showed more binding of the reduced coenzyme (NADH) to GDH and the Lineweaver-Burk plots (with respect to NADH) indicate the existence of substrate inhibition in the presence of octyl glucoside. These observations are aimed at explaining the formation of the molten globule-like structure of GDH, which is induced by a non-ionic detergent such as octyl glucoside.     

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.     

Dihydroorotate dehydrogenase B of Enterococcus faecalis. Characterization and insights into chemical mechanism.

Enterococcus faecalis dihydroorotate dehydrogenase B is a heterodimer of 28 and 33 kDa encoded by the pyrK and pyrDb genes. Both subunits copurify during all chromatographic steps, and, as determined by HPLC, one FMN and one FAD are bound per heterodimer. The enzyme catalyzes efficient oxidation of 4-S-NADH by orotate. Isotope effect and pH data suggest that reduction of flavin by NADH at the PyrK site is only partially rate limiting with no kinetically significant proton transfer occurring in the reductive half-reaction; therefore, a group exhibiting a pK of 5.7 +/- 0.2 represents a residue involved in binding of NADH rather than in catalysis. The reducing equivalents are shuttled between the NADH-oxidizing flavin in PyrK and the orotate-reacting flavin in PyrDb, by iron-sulfur centers through flavin semiquinones as intermediates. A solvent kinetic isotope effect of 2.5 +/- 0.2 on V is indicative of rate-limiting protonation in the oxidative half-reaction and most likely reflects the interaction between the isoalloxazine N1 of the orotate-reducing flavin and Lys 168 (by analogy with L. lactis DHODase A). The oxidative half-reaction is facilitated by deprotonation of the group(s) with pK(s) of 5.8-6.3 and reflects either deprotonation of the reduced flavin or binding of orotate; this step is followed by hydride transfer to C6 and general acid-assisted protonation (pK of 9.1 +/- 0.2) at C5 of the product.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.