Pulsed EPR analysis of tartrate dehydrogenase active-site complexes.

Mn2+.tartrate dehydrogenase.substrate complexes have been examined by electron spin echo envelope modulation spectroscopy. The occurrence of dipolar interactions between Mn2+ and 2H on [2H]pyruvate and [4-2H]NAD(H) confirms that Mn2+ binds at the enzyme active site. The 2H signal arising from labeled pyruvate was lost if the sample was incubated at room temperature, indicating that the enzyme catalyzes exchange between the pyruvate methyl protons and solvent protons. Mn-133Cs dipolar coupling was also observed, which suggests that the monovalent cation cofactor also binds in the active site. The tartrate analogue oxalate was observed to have a significant effect on the binding of NAD(H). Oxalate appears to constrain the binding of NAD(H) so that the nicotinamide portion of the cofactor is held in close proximity to Mn2+. Spectra of enzyme complexes prepared with (R)-[4-2H]NADH showed a more intense 2H signal than analogous complexes prepared with (S)-[4-2H]NADH, demonstrating that the pro-R position of NADH is closer to Mn2+ than the pro-S position and suggesting that tartrate dehydrogenase is an A-side-specific dehydrogenase. Oxalate also affected Cs+ binding; the intensity of the 133Cs signal increased in the presence of oxalate, which suggest that oxalate facilitates binding of Cs+ to the active site or that Cs+ binds closer to Mn2+ when oxalate is present. In addition to signals from substrates, electron spin echo envelope modulation spectra revealed 14N signals that arose from coordination to Mn2+ by nitrogen-containing ligands from the protein; however, the identity of this ligand or ligands remains obscure.     

A transfer nuclear Overhauser effect study of coenzyme binding to distinct sites in binary and ternary complexes in glutamate dehydrogenase

The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxoglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3' and N6 and N2' and N1' to N3' NOE's with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE's. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3'-endo conformation. The absence of evidence for intermolecular NOE's under these conditions indicates that while the active and regulatory NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 A apart.     

Influence of anions and pH on the conformational change of horse liver alcohol dehydrogenase induced by binding of oxidized nicotinamide adenine dinucleotide: binding of chloride to the catalytic metal ion

The conformational change of horse liver alcohol dehydrogenase induced by binding of NAD+ was studied by electronic absorption spectroscopy using cobalt as a spectroscopic probe in the active site. The complex of the enzyme with NAD+ exists in an acidic and an alkaline form. The transition between the two forms proceeds through several intermediates and is controlled by an apparent pKa of 6.9. Only at pH values below this pKa can a complex between enzyme, NAD+, and Cl- be formed. The spectral changes indicate that chloride displaces the cobalt-bound water molecule in a tetracoordinate structure. We conclude that a negative charge at the active site is necessary to stabilize the closed conformation of the enzyme in the presence of NAD+. Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase.     

A shared binding site for NAD+ and coenzyme A in an acetaldehyde dehydrogenase involved in bacterial degradation of aromatic compounds

The meta-cleavage pathway for catechol is a central pathway for the bacterial dissimilation of a wide variety of aromatic compounds, including phenols, methylphenols, naphthalenes, and biphenyls. The last enzyme of the pathway is a bifunctional aldolase/dehydrogenase that converts 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA via acetaldehyde. The structure of the NAD (+)/CoASH-dependent aldehyde dehydrogenase subunit is similar to that of glyceraldehyde-3-phosphate dehydrogenase, with a Rossmann fold-based NAD (+) binding site observed in the NAD (+)-enzyme complex [Manjasetty, B. A., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 6992-6997]. However, the location of the CoASH binding site was not determined. In this study, hydrogen-deuterium exchange experiments, coupled with peptic digest and mass spectrometry, were used to examine cofactor binding. The pattern of hydrogen-deuterium exchange in the presence of CoASH was almost identical to that observed with NAD (+), consistent with the two cofactors sharing a binding site. This is further supported by the observations that either CoASH or NAD (+) is able to elute the enzyme from an NAD (+) affinity column and that preincubation of the enzyme with NAD (+) protects against inactivation by CoASH. Consistent with these data, models of the CoASH complex generated using AUTODOCK showed that the docked conformation of CoASH can fully occupy the cavity containing the enzyme active site, superimposing with the NAD (+) cofactor observed in the X-ray crystal structure. Although CoASH binding Rossmann folds have been described previously, this is the first reported example of a Rossmann fold that can alternately bind CoASH or NAD (+) cofactors required for enzymatic catalysis.     

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

Steady-state kinetics and the inactivation by 2,3-butanedione of the energy-independent transhydrogenase of Escherichia coli cell membranes.

Kinetic measurements indicate that the energy-independent transhydrogenation of 3-acetylpyridine-NAD+ by NADPH in membranes of Escherichia coli follows a rapid equilibrium random bireactant mechanism. Each substrate, although reacting preferentially with its own binding site, is able to interact with the binding site of the other substrate to cause inhibition of enzyme activity. 5'-AMP (and ADP) and 2'-AMP interact with the NAD+- and NADP+-binding sites, respectively. Phenylglyoxal and 2,3-butanedione in borate buffer inhibit transhydrogenase activity presumably by reacting with arginyl residues. Protection against inhibition by 2,3-butanedione is afforded by NADP+, NAD+, and high concentrations of NADPH and NADH. Low concentrations of NADPH and NADH increase the rate of inhibition by 2,3-butanedione. Similar effects are observed for the inactivation of the transhydrogenase by tryptic digestion in the presence of these coenzymes. It is concluded that there are at least two conformations of the active site of the transhydrogenase which differ in the extent to which arginyl residues are accessible to exogenous agents such as trypsin and 2,3-butanedione. One conformation is induced by low concentrations of NADH and NADPH. Under these conditions the coenzymes could be reacting at the active site or at an allosteric site. The stimulation of transhydrogenase activity by low concentrations of the NADH is consistent with the latter possibility.     

Multiple roles of arginine 181 in binding and catalysis in the NAD-malic enzyme from Ascaris suum

The NAD-malic enzyme catalyzes the oxidative decarboxylation of l-malate. Structures of the enzyme indicate that arginine 181 (R181) is within hydrogen bonding distance of the 1-carboxylate of malate in the active site of the enzyme and interacts with the carboxamide side chain of the nicotinamide ring of NADH, but not with NAD+. Data suggested R181 might play a central role in binding and catalysis in malic enzyme, and it was thus changed to lysine and glutamine to probe its potential function. A nearly 100-fold increase in the Km for malate and a 30-fold increase in the Ki for oxalate, an analogue of the enolpyruvate intermediate, in the R181Q and R181K mutants are consistent with a role for R181 in binding substrates. The mutant enzymes also exhibit a >10-fold increase in KiNADH, but only a slight or no change in KNAD, consistent with rotation of the nicotinamide ring into the malate binding site upon reduction of NAD+ to NADH. The activity of the R181Q mutant can be rescued by ammonium ion likely by binding in the pocket vacated by the guanidinium group of R181. Results suggest 2 mol of ammonia bind per mole of active sites with a high-affinity KNH4 of 0.7 +/- 0.1 mM and a low-affinity KNH4 of approximately 420 mM. Occupancy of the high-affinity site, likely by NH4+, results in an increase in the affinity of malate, oxalate, and NADH (with no change in NAD affinity), consistent with the above-proposed roles for R181. The second molecule to bind is likely neutral NH3, and its binding increases V/Et approximately 20-fold. Primary deuterium and 13C isotope effects measured in the absence and presence of ammonium ion suggest R181Q predominantly affects the rate of the reaction by changing the rate of the precatalytic conformational change. The isotope effects do not change upon binding the second mole of ammonia in spite of the 20-fold increase in V/Et. Thus, the R181Q mutant enzyme exists as an equilibrium mixture between active and less active forms, and NH3 stabilizes the more active conformation of the enzyme.     

Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.     

Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site.

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.     

35-Cl nuclear magnetic resonance studies of anion-binding sites in proteins: lactate dehydrogenase.

³⁵Cl nmr relaxation rate measurements have been used to study anion-binding sites in pig heart lactate dehydrogenase. These studies reveal two types of sites, one is intimately associated with the active site, the other is not. The nonactive site has been ascribed to a subunit site in analogy with crystallographic results from the dogfish M₄ enzyme. The binding of either the reduced or the oxidized form of NAD results in an increase in the ³⁵Cl nmr relaxation rate by a factor of 1.8–2. The enhanced nmr relaxation rate of the binary lactate dehydrogenase-NAD complex is reduced on binding of the substrate inhibitor molecules oxamate or oxalate to a value less than that exhibited by lactate dehydrogenase alone. The enhancement of the nmr relaxation rate is attributed to a decrease in the dissociation constant of Cl for the enzyme. The K_p values for Cl binding to the active center site of lactate dehydrogenase is 0.85 m and for lactate dehydrogenase-NADH is 0.25 m. The ratio of these constants, 3.4, agrees well with the measured enhancement value 3.7. The effect of coenzyme analogs on the ³⁵Cl nmr relaxation rate has been examined. 3-Acetylpyridine NAD produces an enhancement of 4.3, thionicotinamide NAD of 2.3, whereas 3-pyridinealdehyde, adenosinediphosphoribose, and adenosine diphosphate do not affect the nmr relaxation state of Cl bound to lactate dehydrogenase.     

Role of residues in the adenosine binding site of NAD of the Ascaris suum malic enzyme

Ascaris suum mitochondrial malic enzyme catalyzes the divalent metal ion dependent conversion of l-malate to pyruvate and CO(2), with concomitant reduction of NAD(P) to NAD(P)H. In this study, some of the residues that form the adenosine binding site of NAD were mutated to determine their role in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the K(m) for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/E(t) decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/E(t), contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.     

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

Crystal structures of transhydrogenase domain I with and without bound NADH

Transhydrogenase (TH) is a dimeric integral membrane enzyme in mitochondria and prokaryotes that couples proton translocation across a membrane with hydride transfer between NAD(H) and NADP(H) in soluble domains. Crystal structures of the NAD(H) binding alpha1 subunit (domain I) of Rhodospirillum rubrum TH have been determined at 1.8 A resolution in the absence of dinucleotide and at 1.9 A resolution with NADH bound. Each structure contains two domain I dimers in the asymmetric unit (AB and CD); the dimers are intimately associated and related by noncrystallographic 2-fold axes. NADH binds to subunits A and D, consistent with the half-of-the-sites reactivity of the enzyme. The conformation of NADH in subunits A and D is very similar; the nicotinamide is in the anti conformation, the A-face is exposed to solvent, and both N7 and O7 participate in hydrogen bonds. Comparison of subunits A and D to six independent copies of the subunit without bound NADH reveals multiple conformations for residues and loops surrounding the NADH site, indicating flexibility for binding and release of the substrate (product). The NADH-bound structure is also compared to the structures of R. rubrum domain I with NAD bound (PDB code 1F8G) and with NAD bound in complex with domain III of TH (PDB code 1HZZ). The NADH- vs NAD-bound domain I structures reveal conformational differences in conserved residues in the NAD(H) binding site and in dinucleotide conformation that are correlated with the net charge, i.e., oxidation state, of the nicotinamides. The comparisons illustrate how nicotinamide oxidation state can affect the domain I conformation, which is relevant to the hydride transfer step of the overall reaction.     

Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes.

The conformation of NAD bound to diphtheria toxin (DT), an ADP-ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)-binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N-glycosidic bond between the nicotinamide ring and the N-ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase-bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT''s function as an ADP-ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle chi N approximately 0 degree about the scissile N-glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle chi N approximately 0 degree could contribute to catalysis, possibly by orienting the C1''N atom of NAD for nucleophilic attack, or by placing strain on the N-glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)-binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel beta-sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C-terminal end of a parallel beta-sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP-ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP-ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.     

3-磷酸甘油醛脱氢酶的胍变性——全位与半位修饰酶的比较

酵母3-磷酸甘油醛脱氢酶在盐酸胍溶液中内源荧光及NAD荧光衍生物的410nm特征荧光发射光谱的变化结果提示,全位及半位修饰羧甲基酶活性部位与NAD共价连接的荧光衍生物的形成,明显受到盐酸胍的干扰,并且前者比后者更为显著.全位及半位修饰光照酶的特征荧光在低胍浓度下较内源荧光降低更为显著,同时伴有最大发射峰先红移后兰移的现象.NAD荧光衍生物的特征荧光在胍溶液中减弱的动力学过程分为快相和慢相,快相一级动力学常数比慢相的大两个数量级,全位及半位修饰酶的特征荧光的减弱快慢相速度常数分别属于同一个数量级.以上结果提示:酶活性部位的构象较整个分子来说更易被变性剂扰乱,柔性强于整个分子;NAD荧光衍生物的形成需要活性部位具有正确的空间几何结构.     

A kinetic alignment of orthologous inosine-5'-monophosphate dehydrogenases

IMP dehydrogenase (IMPDH) catalyzes two very different chemical transformations, a dehydrogenase reaction and a hydrolysis reaction. The enzyme toggles between the open conformation required for the dehydrogenase reaction and the closed conformation of the hydrolase reaction by moving a mobile flap into the NAD site. Despite these multiple functional constraints, the residues of the flap and NAD site are highly diverged, and the equilibrium between open and closed conformations ( K c ) varies widely. In order to understand how differences in the dynamic properties of the flap influence the catalytic cycle, we have delineated the kinetic mechanism of IMPDH from the pathogenic protozoan parasite Cryptosporidium parvum ( CpIMPDH), which was obtained from a bacterial source through horizontal gene transfer, and its host counterpart, human IMPDH type 2 (hIMPDH2). Interestingly, the intrinsic binding energy of NAD (+) differentially distributes across the dinucleotide binding sites of these two enzymes as well as in the previously characterized IMPDH from Tritrichomonas foetus ( TfIMPDH). Both the dehydrogenase and hydrolase reactions display significant differences in the host and parasite enzymes, in keeping with the phylogenetic and structural divergence of their active sites. Despite large differences in K c , the catalytic power of both the dehydrogenase and hydrolase conformations are similar in CpIMPDH and TfIMPDH. This observation suggests that the closure of the flap simply sets the stage for catalysis rather than plays a more active role in the chemical transformation. This work provides the essential mechanistic framework for drug discovery.     

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.     

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.