Human glutathione-dependent formaldehyde dehydrogenase. Structures of apo,binary, and inhibitory ternary complexes

The human glutathione-dependent formaldehyde dehydrogenase is unique among the structurally studied members of the alcohol dehydrogenase family in that it follows a random bi bi kinetic mechanism. The structures of an apo form of the enzyme, a binary complex with substrate 12-hydroxydodecanoic acid, and a ternary complex with NAD+ and the inhibitor dodecanoic acid were determined at 2.0, 2.3, and 2.3 A resolution by X-ray crystallography using the anomalous diffraction signal of zinc. The structures of the enzyme and its binary complex with the primary alcohol substrate, 12-hydroxydodecanoic acid, and the previously reported binary complex with the coenzyme show that the binding of the first substrate (alcohol or coenzyme) causes only minor changes to the overall structure of the enzyme. This is consistent with the random mechanism of the enzyme where either of the substrates binds to the free enzyme. The catalytic-domain position in these structures is intermediate to the "closed" and "open" conformations observed in class I alcohol dehydrogenases. More importantly, two different tetrahedral coordination environments of the active site zinc are observed in these structures. In the apoenzyme, the active site zinc is coordinated to Cys44, His66 and Cys173, and a water molecule. In the inhibitor complex, the coordination environment involves Glu67 instead of the solvent water molecule. The coordination environment involving Glu67 as the fourth ligand likely represents an intermediate step during ligand exchange at the active site zinc. These observations provide new insight into metal-assisted catalysis and substrate binding in glutathione-dependent formaldehyde dehydrogenase.     

The reaction of bovine glutamate dehydrogenase with periodate-oxidised ADP

1. The reactive analogue oADP produced by periodate oxidation of ADP has been studied as a potential affinity label for the enzyme bovine glutamate dehydrogenase, using circular dichroism (CD) difference spectroscopy to monitor specific binding. 2. The analogue binds stoichiometrically, rapidly and reversibly to the adenine nucleotide binding site with Kd approximately equal to 12 microM (20 degrees C, pH 7) with characteristic intensification of the adenine nucleotide CD at 260 nm. 3. This complex is unstable and decays with a half-life of about 1.5 h; the analogue then becomes attached as a Schiff base to a number of subsidiary sites, including the enzyme active site, with partial inactivation of the enzyme. 4. Depending upon initial concentration of oADP, the enzyme activity is progressively lost during the slow reaction; following borohydride reduction, up to four molecules of analogue are bound/subunit. 5. Protection against loss of enzyme activity is afforded by the coenzyme NAD+ plus glutarate or L-hydroxyglutarate (an effective inhibitor), or by glutarate alone, but not by NAD+ alone. 6. Spectroscopic and protection studies indicate that after the decay of the specific CD signal, the enzyme retains the capacity to bind ADP, but that this is progressively lost in parallel with decay of enzymic activity. 7. The results are consistent with proximity or functional interaction between the adenine nucleotide site and the coenzyme binding portion of the active site. 8. Thus oADP does not act as a true affinity label for the adenine nucleotide binding site, but the reaction subsequent to binding at that site shows some specificity directed towards the active site.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.     

Structural and functional consequences of coenzyme binding to the inactive asian variant of mitochondrial aldehyde dehydrogenase: roles of residues 475 and 487

The common mitochondrial aldehyde dehydrogenase (ALDH2) ALDH2(*)2 polymorphism is associated with impaired ethanol metabolism and decreased efficacy of nitroglycerin treatment. These physiological effects are due to the substitution of Lys for Glu-487 that reduces the k(cat) for these processes and increases the K(m) for NAD(+), as compared with ALDH2. In this study, we sought to understand the nature of the interactions that give rise to the loss of structural integrity and low activity in ALDH2(*)2 even when complexed with coenzyme. Consequently, we have solved the crystal structure of ALDH2(*)2 complexed with coenzyme to 2.5A(.) We have also solved the structures of a mutated form of ALDH2 where Arg-475 is replaced by Gln (R475Q). The structural and functional properties of the R475Q enzyme are intermediate between those of wild-type and the ALDH2(*)2 enzymes. In both cases, the binding of coenzyme restores most of the structural deficits observed in the apoenzyme structures. The binding of coenzyme to the R475Q enzyme restores its structure and catalytic properties to near wild-type levels. In contrast, the disordered helix within the coenzyme binding pocket of ALDH2(*)2 is reordered, but the active site is only partially reordered. Consistent with the structural data, ALDH2(*)2 showed a concentration-dependent increase in esterase activity and nitroglycerin reductase activity upon addition of coenzyme, but the levels of activity do not approach those of the wild-type enzyme or that of the R475Q enzyme. The data presented shows that Glu-487 maintains a critical function in linking the structure of the coenzyme-binding site to that of the active site through its interactions with Arg-264 and Arg-475, and in doing so, creates the stable structural scaffold conducive to catalysis.     

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.     

The nickel enzyme methyl-coenzyme M reductase from methanogenic archaea: in vitro interconversions among the EPR detectable MCR-red1 and MCR-red2 states

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme contains tightly bound the nickel porphinoid F430. The nickel enzyme has been shown to be active only when its prosthetic group is in the Ni(I) reduced state. In this state MCR exhibits the nickel-based EPR signal red1. We report here for the MCR from Methanothermobacter marburgensis that the EPR spectrum of the active enzyme changed upon addition or removal of coenzyme M, methyl coenzyme M and/or coenzyme B. In the presence of methyl-coenzyme M the red1 signal showed a more resolved 14N-superhyperfine splitting than in the presence of coenzyme M indicating a possible axial ligation of the substrate to the Ni(I). In the presence of methyl-coenzyme M and coenzyme B the red1 signal was the same as in the presence of methyl-coenzyme M alone. However, in the presence of coenzyme M and coenzyme B a highly rhombic EPR signal, MCR-red2, was induced, which was found to be light sensitive and appeared to be formed at the expense of the MCR-red1 signal. Upon addition of methyl-coenzyme M, the red2 signal disappeared and the red1 signal increased again. The red2 signal of MCR with 61Ni-labeled cofactor was significantly broadened indicating that the signal is nickel or nickel-ligand based.     

Electrostatic field effects of coenzymes on ligand binding to catalytic zinc in liver alcohol dehydrogenase

The synergism between coenzyme and anion binding to liver alcohol dehydrogenase has been examined by equilibrium measurements and transient-state kinetic methods to characterize electrostatic interactions of coenzymes with ligands which are bound to the catalytic zinc ion of the enzyme subunit. Inorganic anions typically exhibit an at least 200-fold higher affinity for the general anion-binding site than for catalytic zinc on complex formation with free enzyme. Acetate and SCN- interact more strongly with catalytic zinc in the enzyme X NAD+ complex than with the general anion-binding site in free enzyme. CN- shows no significant affinity for the general anion-binding site, but combines to catalytic zinc in the absence as well as the presence of coenzymes. Coordination of CN- to catalytic zinc weakens the binding of NADH by a factor of 50, and tightens the binding of NAD+ to approximately the same extent through interactions which do not include any contributions from covalent adduct formation between CN- and NAD+. These observations provide unambiguous information about the magnitude of electrostatic field effects of coenzymes on anion (e.g. hydroxyl ion) binding to catalytic zinc. They lead to the important inference that coenzyme binding must be strongly affected by ionization of zinc-bound water irrespective of the actual acidity of the latter group. It is concluded on such grounds that the much debated pH dependence of coenzyme binding to liver alcohol dehydrogenase must derive from ionization of zinc-bound water. The assumption that such is not the case leads to the inference that there is no detectable effect of ionization of zinc-bound water on coenzyme binding over the pH range 6-12, a possibility which is definitely excluded by the present results.     

Electrostatic effect upon association of reduced nicotinamide adenine dinucleotide and equine liver alcohol dehydrogenase

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray analysis of structural changes induced by reduced nicotinamide adenine dinucleotide when bound to cysteine-46-carboxymethylated liver alcohol dehydrogenase

The structure of the complex between Cys-46-carboxymethylated horse liver alcohol dehydrogenase (CM-LADH) and reduced nicotinamide adenine dinucleotide (NADH) has been determined by X-ray analysis. The complex represents NADH binding to the orthorhombic, "open" conformation of the enzyme. Coenzyme binding here induces a local structural change in the peptide loop 293-297, but there is no domain rotation, as observed for the "closed" conformation of the protein. This local movement of a few residues in the loop is sufficient to trap the nicotinamide ring of NADH within the active-site area close to a productive binding position. The carboxymethyl group on the zinc ligand cysteine-46 is oriented between the pyrophosphate bridge of NADH and the guanidinium group of arginine-369 and can occupy this position because the coenzyme binding cleft remains open and unchanged upon coenzyme binding. The zinc coordination sphere is distorted, and the position of the metal atom is shifted 1 A compared to native unliganded LADH. The distance between the zinc ion and the sulfur of the alkylated cysteine residue is of the order of 3 A. Alkylation experiments were performed at 0.15 and 10 mM iodoacetate, and peptide maps were examined. Gentle treatment with reagent yields an enzyme product which is substituted at only one of the two zinc binding sites per subunit of LADH (Cys-46). This enzyme species maintains its structural integrity; it binds coenzyme which induces conformational changes resolved into two steps. Thus, in addition to the orthorhombic complex, a crystalline NADH complex in the closed conformation of CM-LADH was obtained. These crystals showed enzymic activity, and single crystals were analyzed with microspectrophotometric methods. Formation of the stable crystalline abortive complex between CM-LADH-NAD+ and 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) could be observed upon addition of excess aldehyde to the closed complex of CM-LADH-NADH. The CM-LADH-NAD+-DACA complex is characterized by an intense absorption band with a lambda max at 456 nm which corresponds to a shift in the spectrum of free DACA of approximately 60 nm. At the higher concentration of iodoacetate, three of the cysteine ligands to the second zinc atom (Cys-100, -103, and -111) are alkylated in addition to Cys-46. This enzyme product rapidly denatures and cannot be crystallized under our conditions. This is an experimental indication that the intact noncatalytic zinc binding site contributes to the structural stability of the protein.     

Brain succinic semialdehyde dehydrogenase. Reactions of sulfhydryl residues connected with catalytic activity.

Incubation of an NAD+-dependent succinic semialdehyde dehydrogenase from bovine brain with 4-dimethylaminoazobenzene-4-iodoacetamide (DABIA) resulted in a time-dependent loss of enzymatic activity. This inactivation followed pseudo first-order kinetics with a second-order rate constant of 168 m(-1).min(-1). The spectrum of DABIA-labeled enzyme showed a characteristic peak of the DABIA alkylated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and the loss of enzyme activity, which extrapolates to a stoichiometry of 8.0 mol DABIA derivatives per mol enzyme tetramer. This inactivation was prevented by preincubating the enzyme with substrate, succinic semialdehyde, but not by preincubating with coenzyme NAD+. After tryptic digestion of the enzyme modified with DABIA, two peptides absorbing at 436 nm were isolated by reverse-phase HPLC. The amino acid sequences of the DABIA-labeled peptides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively. These sites are identical to the putative active site sequences of other brain succinic semialdehyde dehydrogenases. These results suggest that the catalytic function of succinic semialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site.     

Carboxymethylation of horse-liver alcohol dehydrogenase in the crystalline state. The active-site zinc region and general anion-binding site of the enzyme correlated in primary and teritiary structures.

Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)2-/4 , 1,10-phenanthroline, Au(CN)-/2 and AMP also prevented alkylation with decreasing effectiveness. Crystallographic studies of the alkylated enzyme show that the carboyxmethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN2-/4 to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom. The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN2-/4 and to a lesser extent Au(CN)-/2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc. Identificationof this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.     

Substitutions in a flexible loop of horse liver alcohol dehydrogenase hinder the conformational change and unmask hydrogen transfer.

When horse liver alcohol dehydrogenase binds coenzyme, a rotation of about 10 degrees brings the catalytic domain closer to the coenzyme binding domain and closes the active site cleft. The conformational change requires that a flexible loop containing residues 293-298 in the coenzyme binding domain rearranges so that the coenzyme and some amino acid residues from the catalytic domain can be accommodated. The change appears to control the rate of dissociation of the coenzyme and to be necessary for installation of the proton relay system. In this study, directed mutagenesis produced the activated Gly293Ala/Pro295Thr enzyme. X-ray crystallography shows that the conformations of both free and complexed forms of the mutated enzyme and wild-type apoenzyme are very similar. Binding of NAD(+) and 2,2, 2-trifluoroethanol do not cause the conformational change, but the nicotinamide ribose moiety and alcohol are not in a fixed position. Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations. The mutated enzyme may be active in the "open" conformation. The turnover numbers with ethanol and acetaldehyde increase 1.5- and 5.5-fold, respectively, and dissociation constants for coenzymes and other kinetic constants increase 40-2,000-fold compared to those of the native enzyme. Substrate deuterium isotope effects on the steady state V or V/K(m) parameters of 4-6 with ethanol or benzyl alcohol indicate that hydrogen transfer is a major rate-limiting step in catalysis. Steady state oxidation of benzyl alcohol is most rapid above a pK of about 9 for V and V/K(m) and is 2-fold faster in D(2)O than in H(2)O. The results are consistent with hydride transfer from a ground state zinc alkoxide that forms a low-barrier hydrogen bond with the hydroxyl group of Ser48.     

Methyl-coenzyme M formation in methanogenic archaea. Involvement of zinc in coenzyme M activation.

Methyl-coenzyme M (2-methylthioethane sulfonate) is the key intermediate of methane formation in methanogenic archaea. It is generated from coenzyme M (2-mercaptoethane sulfonate) in methyl transfer reactions catalyzed by proteins containing zinc. Here, we report that, for methyltransferase MtaA from Methanosarcina barkeri, the zinc is involved in coenzyme M activation. For the experiments an inactive MtaA apoprotein was obtained by heterologous overproduction in Escherichia coli grown in the presence of 2 mM EDTA. The apoprotein was found to react with zinc or cobalt to the fully active holoenzyme. Appoximately 1 mol of transition metal was bound per mol of protein. Upon incubation of the holoenzyme with coenzyme M approximately 1 mol of proton was released per mol of zinc or cobalt. Protons were not released upon incubation of the apoprotein with coenzyme M or of the holoprotein with other thiol compounds or with methyl-coenzyme M. The findings are interpreted as indicating that the role of the transition metal in MtaA is to lower the microscopic pKa of the thiol group of coenzyme M by coordination to the zinc, and thus to increase its nucleophilicity for methyl group attack. The pKZn2+ of MtaA was re-determined and found to be > 15 and not 9.6 as previously reported by us.     

Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant

Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.     

Inactivation of monoamine oxidase by allylamine does not result in flavin attachment

[1-3H]Allylamine was synthesized by sodium boro[3H]hydride reduction of acrolein followed by direct conversion of the [1-3H]allyl alcohol to N-allylphthalimide with triphenylphosphine, diethylazodicarboxylate, and phthalimide. The protecting group was removed with hydrazine. Inactivation of beef liver mitochondrial monoamine oxidase with [1-3H]allylamine led to incorporation of 1-6 eq of inactivator/active site depending upon the length of incubation time. Inactivation and radioactivity incorporation coincided; however, after 1 eq of tritium was incorporated and 5% enzyme activity remained, additional radioactivity continued to become incorporated into the enzyme. The optical spectrum of the FAD coenzyme changed during inactivation from that of oxidized to reduced flavin. Following dialysis of the inactivated enzyme, the spectrum remained reduced, but denaturation in urea rapidly resulted in reoxidation of the flavin. Under these same denaturing conditions, 96% of the radioactivity associated with the enzyme remained bound, therefore indicating that allylamine attachment is not to the flavin coenzyme but rather to an active site amino acid residue. The adduct also was stable to base and, to a lesser degree, acid treatment. Although allylamine and N-cyclopropylbenzylamine appear to be oxidized by monoamine oxidase to give 3-(amino acid residue) propanal adducts, two different amino acids seem to be involved because of a difference in stability of the adducts. The mechanisms for inactivation of monoamine oxidase by allylamine and reactivation by benzylamine are discussed in relation to previously reported results.     

Structural differences between apo- and holoenzyme of horse liver alcohol dehydrogenase.

The three-dimensional structure of a ternary complex of horse liver alcohol dehydrogenase with reduced nicotinamide adenine dinucleotide and the inhibitor dimethyl sulfoxide has been determined to 4.5 A resolution independently of the apoenzyme structure. The electron density maps of both structures have been compared. The two coenzyme binding domains which form the center of the dimer molecular have retained their conformation and orientation within the molecule whereas the catalytic domains rotate and narrow the cleft between the domains. The active site becomes shielded from the solution by a combination of this rotation, local movements of a loop from residues 53 to 57 and coenzyme and substrate binding. Both subunits bind coenzyme and inhibitor to the same extent. The nicotinamide ring of the coenzyme is positioned close to the active zinc atom and the inhibitor is bound to this zinc atom. The difference between the two crystallographically independent subunits is small. The proposed mechanisms of action for the enzyme based on the apoenzyme structure are confirmed by the present investigation.     

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: Optical spectra of binary and ternary complexes with coenzymes,coenzyme analogues,substrates, and inhibitors

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M^?1 cm^?1, 407 nm (3500 M^?1 cm^?1), 505 nm (300 M^?1 cm^?1), 570 nm (?130 M^?1 cm^?1), and 680 nm (?80 M^?1 cm^?1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry.The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH > 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal ion which acts as a Lewis acid in both substrate coordination and catalysis.     

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH [( S]NADPH). For native enzyme, ultrafiltration studies revealed that [S]NADPH reversibly occupies about two sites/enzyme subunit in the absence of other ligands; by the addition of ADP, [S]NADPH binding can be limited to one molecule/subunit. The enzyme is irreversibly inactivated by reaction with 4-(iodoacetamido)salicylic acid (ISA) at lysine126 within the 2-oxoglutarate binding site [Holbrook, J.J., Roberts, P.A. & Wallis, R.B. (1973) Biochem. J. 133, 165-171]. ISA-modified enzyme binds 1 molecule [S]NADPH/subunit in the absence of ADP, suggesting that reaction at the substrate site blocks binding at the catalytic, but not at the regulatory site. The fluorescence spectrum of ISA-modified enzyme overlaps the absorption spectrum of [S]NADPH allowing a distance measurement between these sites by resonance energy transfer. [S]NADPH quenches the emission of ISA-modified enzyme, yielding 3.2 nm as the average distance between sites. ADP competes for the [S]NADPH site but does not affect the fluorescence of ISA-modified enzyme, indicating that [S]NADPH quenching is attributable to energy transfer rather than to a conformational change. The 3.2 nm thus represents the distance between the 2-oxoglutarate and reduced coenzyme regulatory sites of glutamate dehydrogenase.     

Circular dichroism studies of the coenzyme environment in the active sites of mutant forms of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex.

The circular dichroism has been used to evaluate the effect of mutation on the environment of the pyridoxal phosphate coenzyme in the active site of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Seven mutant forms of the alpha 2 beta 2-complex with single amino acid replacements at residues 87, 109, 188, 306, and 350 of the beta-subunit have been prepared by site-directed mutagenesis, purified to homogeneity, and characterized by absorption and circular dichroism spectroscopy. Since the wild type and mutant alpha 2 beta 2 complexes all exhibit positive circular dichroism in the coenzyme absorption band, pyridoxal phosphate must bind asymmetrically in the active site of these enzymes. However, the coenzyme may have an altered orientation or active site environment in five of the mutant enzymes that display less intense ellipticity bands. The mutant enzyme in which lysine 87 is replaced by threonine has very weak ellipticity at 400 nm. Since lysine 87 forms a Schiff base with pyridoxal phosphate in the wild type enzyme, our results demonstrate the importance of the Schiff base linkage for rigid or asymmetric binding. Although the mutant enzymes display spectra in the presence of L-serine that differ from that of the wild type enzyme, addition of alpha-glycerol 3-phosphate converts the spectra of two of the mutant enzymes to that of the wild type enzyme. We conclude that this alpha-subunit ligand may produce a conformational change in the alpha-subunit that is transmitted to the mutant beta-subunits and partially corrects conformational alterations in the mutant enzymes.     

Functional arginine residues involved in coenzyme binding by dihydrofolate reductase

Reaction of dihydrofolate reductase from amethopterin-resistant $\text{Lactobacillus}\text{casei}$ with phenylglyoxal results in a complete loss of enzyme activity. This inactivation is concomitant with the modification of five of a total of eight arginine residues per mole of enzyme. In the presence of the reduced coenzyme, NADPH, two of the five reactive arginines are protected from chemical modification with complete retention of enzyme activity. The results suggest the involvement of essential arginine residues at or near the coenzyme binding site and thus at or near the active center of the enzyme.