Proton nuclear magnetic resonance studies of the effects of ligand binding on tryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei

We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been assigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.     

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex

The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.     

Dihydrofolate reductase: control of the mode of substrate binding by aspartate 26

The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent and which differ in the orientation of the pteridine ring of the substrate in the binding site. The Asp26----Asn mutant of L. casei dihydrofolate reductase has been prepared by oligonucleotide-directed mutagenesis and studied by one- and two-dimensional 1H-NMR spectroscopy. NMR studies of the mutant enzyme--folate--NADP+ complex show that this exists to greater than 90% in a single conformation over the pH* range 5-7.1. The single conformation observed corresponds to conformation I (the 'methotrexate-like' conformation) of the wild-type enzyme--folate--NADP+ complex. These observations demonstrate that Asp26 is the ionizable group controlling the pH-dependence of the conformational equilibrium seen in the wild-type enzyme.     

Characterization of rates of ring-flipping in trimethoprim in its ternary complexes with Lactobacillus casei dihydrofolate reductase and coenzyme analogues

NMR measurements have been used to investigate rates of ring-flipping and the activation parameters for the trimethoxybenzyl ring of the antibacterial drug trimethoprim (TMP) bound to Lactobacillus casei dihydrofolate reductase (DHFR) for a series of ternary complexes formed with analogues of the coenzyme NADPH. Rates were obtained at several temperatures from line shape analyses ((13)C-edited HSQC (1)H spectra) and transfer of magnetization measurements (zz-HSQC) on complexes containing 3'-O-[(13)C]trimethoprim. Examination of the structures of the complexes indicates that ring-flipping can only be achieved following major conformational changes and transient fluctuations of the protein and coenzyme structure around the trimethoxybenzyl ring. There is no simple correlation between rates of ring-flipping and binding constants. The presence of the coenzyme nicotinamide ring (in either its reduced or its oxidized forms) in the binding site close to the trimethoxybenzyl ring moiety is the major factor reducing the ring-flipping on coenzyme binding. Thus, the ternary complex with NADPH shows the largest reduction in the rate of ring-flipping (11 +/- 3 s(-)(1) at 298 K) as compared with the binary complex (793 +/- 80 s(-)(1) at 298 K). Complexes with NADPH analogues that either have no nicotinamide ring or are known to have their nicotinamide rings removed from the binding site show the smallest reductions. For the DHFR.TMP.NADP(+) complex where there are two conformations present, very different rates of ring-flipping were observed for the two forms. The activation parameters (DeltaH() and DeltaS()) for the ring-flipping in all the complexes are discussed in terms of the protein-ligand interactions and the possible constraints on the pathway through the transition state.     

Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.     

Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme

The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.     

Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode

Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) is a NADPH-dependent enzyme unrelated to chromosomal DHFR in amino acid sequence and structure. R67 DHFR is insensitive to the bacterial drug trimethoprim in contrast to chromosomal DHFR. The crystal structure of Q67H mutant of R67 DHFR bound to NADP(+) has been determined at 1.15 angstroms resolution. The cofactor assumes an extended conformation with the nicotinamide ring bound near the center of the active site pore, the ribose and pyrophosphate group (PP(i)) extending toward the outer pore. The ribonicotinamide exhibits anti conformation as in chromosomal DHFR complexes. The relative orientation between the PP(i) and the nicotinamide ribose differs from that observed in chromosomal DHFR-NADP(+) complexes. The coenzyme displays symmetrical binding mode with several water-mediated hydrogen bonds with the protein besides ionic, stacking, and van der Waals interactions. The structure provides a molecular basis for the observed stoichiometry and cooperativity in ligand binding. The ternary model based on the present structure and the previous R67 DHFR-folate complex provides insight into the catalytic mechanism and indicates that the relative orientation of the reactants in plasmid DHFR is different from that seen in chromosomal DHFRs.     

Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase

Amino acid residues Thr-178, Val-203, and Val-292, which interact with the nicotinamide ring of the coenzyme bound to alcohol dehydrogenase (ADH), may facilitate hydride transfer and hydrogen tunneling by orientation and dynamic effects. The T178S, T178V, V203A, V292A, V292S, and V292T substitutions significantly alter the steady state and transient kinetics of the enzyme. The V292A, V292S, and V292T enzymes have decreased affinity for coenzyme (NAD+ by 30-50-fold and NADH by 35-75-fold) as compared to the wild-type enzyme. The substitutions in the nicotinamide binding site decrease the rate constant of hydride transfer for benzyl alcohol oxidation by 3-fold (for V292T ADH) to 16-fold (for V203A ADH). The modest effects suggest that catalysis does not depend critically on individual residues and that several residues in the nicotinamide binding site contribute to catalysis. The structures of the V292T ADH-NAD+-pyrazole and wild-type ADH-NAD+-4-iodopyrazole ternary complexes are very similar. Only subtle changes in the V292T enzyme cause the large changes in coenzyme binding and the small change in hydride transfer. In these complexes, one pyrazole nitrogen binds to the catalytic zinc, and the other nitrogen forms a partial covalent bond with C4 of the nicotinamide ring, which adopts a boat conformation that is postulated to be relevant for hydride transfer. The results provide an experimental basis for evaluating the contributions of dynamics to hydride transfer.     

Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

Amino acids in three highly conserved segments of the Ca2(+)-ATPase. Asp-Pro-Pro-Arg604, Thr-Gly-Asp627, Thr-Gly-Asp703 as well as Asp707, have been proposed to participate in formation of the nucleotide binding site. We have tested this hypothesis by investigating the properties of mutants with alterations to amino acids within these segments. Most of the mutants were found to be defective in Ca2+ transport function. The inactive mutants could be separated into two classes on the basis of the kinetics of phosphoenzyme intermediate formation and decomposition. One group, Asp601----Asn, Pro603----Leu, Asp627----Glu, and Asp703----Asn, formed phosphoenzyme intermediates with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that both the high affinity Ca2+ binding sites and the nucleotide binding sites were intact. In each of these mutants, however, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive phosphoenzyme intermediate very slowly, relative to the wild-type enzyme. Thus the inability of these mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. Another group, Asp601----Glu, Pro603----Gly, Asp707----Glu, and Asp707----Asn, did not form detectable phosphoenzyme intermediates from either ATP or Pi. Although we have demonstrated an effect on Ca2+ transport of mutations in each of the highly conserved regions predicted to be involved in ATP binding, we cannot yet define their roles in ATP-dependent Ca2+ transport.     

Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a 13C NMR study using selectively 13C-enriched trimethoprim

We have measured the 13C chemical shifts for trimethoprim molecules selectively enriched with 13C at the 2-, 4-, 5-, 6-, and 7-positions and the p-OCH3 position in their complexes with Lactobacillus casei dihydrofolate reductase in the presence and absence of coenzyme analogues. The C2 carbon shifts indicate that the pyrimidine ring is protonated at N1 in all the complexes of trimethoprim with the enzyme and coenzymes and in each case the pyrimidine ring is binding in a similar way to that of the corresponding part of methotrexate in the enzyme-methotrexate complex. The C6 carbon of trimethoprim shows a large upfield shift in all complexes (3.51 to 4.70 ppm) but no shift in the complex of 2,4-diaminopyrimidine with the enzyme: these shifts probably arise from steric interactions between the C1' and C2' carbons and the H6 proton, which approach van der Waals contact in the folded conformation adopted by trimethoprim when bound to the enzyme. The large shift observed for C6 in all complexes indicates that the basic folded conformation is present in all of them. A comparison of the 13C shifts in the enzyme-trimethoprim-NADPH complex with those in the enzyme-trimethoprim binary complex shows substantial changes even for carbons such as C6 and p-OCH3 (0.46 and -0.36 ppm, respectively), which are remote from the coenzyme: these are caused by ligand-induced conformational changes that may involve displacement of the helix containing residues 42-49.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Investigating interdomain region mutants Phe194----Leu and Phe194----Trp of yeast phosphoglycerate kinase by 1H-NMR spectroscopy.

Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H-NMR spectroscopy, it was found that the mutations at position 194 induce both local and long-range conformational changes in the protein. It was also found that 3-phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild-type phosphoglycerate kinase. The affinity of mutant Phe194----Trp for 3-phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194----Leu mutant is reduced by about threefold relative to the wild-type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194----Leu mutant when compared to wild-type or Phe194----Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194----Leu mutant the Km values for both 3-phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild-type enzyme, while for mutant Phe194----Trp, the Km values for 3-phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild-type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194----Trp and less for mutant Phe194----Leu, relative to that observed for wild-type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3-phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3-phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.     

Use of transferred nuclear Overhauser effect measurements to compare binding of coenzyme analogues to dihydrofolate reductase

Transferred nuclear Overhauser effect measurements have been made on complexes of NADP+ and thioNADP+ with Lactobacillus casei dihydrofolate reductase to provide information about the glycosidic bond conformations in these complexes. Both NADP+ and thioNADP+ are shown to have very similar anti conformations about their adenine glycosidic bonds when bound to the enzyme. However, their nicotinamide glycosidic bond conformations are very different: while NADP+ binds in an exclusively anti conformation, thioNADP+ binds with a distribution of syn/anti conformations very similar to that observed in nicotinamide mononucleotides in free solution (approximately 50:50). Thus for thioNADP+, binding to the enzyme does not significantly perturb the potential function for rotation about the nicotinamide glycosidic bond. Earlier NMR studies [Hyde, E. I., Birdsall, B., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1980) Biochemistry 19, 3738] had indicated that large downfield 1H shifts of the nicotinamide ring protons (0.61-1.36 ppm) are detected on binding NADP+ while only very small shifts (less than 0.1 ppm) are observed in complexes with thioNADP+. The chemical shift and conformational findings are best explained if the thionicotinamide ring extends into solution making essentially no contacts with the enzyme.     

Dihydrofolate reductase from Lactobacillus casei. Stereochemistry of NADPH binding.

The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.     

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.     

The interaction of adenine with its binding site in rabbit muscle glyceraldehyde 3-phosphate dehydrogenase studied by fluorescence decay.

The fluorescence decay mechanism of 1, N⁶-ethenoadenosine diphosphoribose bound to rabbit muscle glyceraldehyde 3-phosphate dehydrogenase markedly differs from that of the intact coenzyme analog (εNAD⁺) bound to the same enzyme. In the latter case the fluorescence is partially quenched by interactions between the ethenoadenine ring and amino acid residues in its binding site. Binding of the nicotinamide moiety of the coenzyme thus affects the relative orientation of the adenine ring within its binding site leading to the quenching interactions. The interactions of the adenine group with its binding site induce conformational changes in the enzyme which affect the binding of additional coenzyme molecules. The nicotinamide base thus determines, indirectly, the negative cooperativity found in NAD⁺ binding.     

Trimethoprim binding to bacterial and mammalian dihydrofolate reductase: a comparison by proton and carbon-13 nuclear magnetic resonance

The binding of trimethoprim to dihydrofolate reductase from L1210 mouse lymphoma cells has been studied by measuring the changes in chemical shift of nuclei of the ligand that accompanying binding. The 6- and 2',6'-proton chemical shifts of bound trimethoprim have been determined by transfer of saturation experiments, and the 2-carbon chemical shift has been determined by using [2-13C]trimethoprim. The changes in proton chemical shift are substantially smaller than those accompanying binding to bacterial dihydrofolate reductase [Cayley, P. J., Albrand, J. P., Feeney, J., Robert, G. C. K., Piper, E. A., & Burgen, A. S. V. (1979) Biochemistry 18, 3886]. It is shown that this difference arises largely from the fact that trimethoprim adopts different conformations when bound to mammalian and to bacterial dihydrofolate reductase. The proton chemical shifts are interpreted in terms of ring-current contributions from the two aromatic rings of trimethoprim itself and the nearby aromatic amino acid residues of the enzyme. The latter have been located by using the refined crystallographic coordinates of the Lactobacillus casei and Escherichia coli reductases in their complexes with methotrexate [Bolin, J. T., Filman, D. J., Matthews, D. A. & Kraut, J. (1982) J. Biol. Chem. 257, 13650], under the assumption that, as indicated by the 13C chemical shifts, the diaminopyrimidine ring of trimethoprim binds in the same way as does the corresponding part of methotrexate. With use of these assumptions, the conformation of trimethoprim bound to the dihydrofolate reductases from L. casei, E. coli, and L1210 cells has been calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of human aldose reductase holoenzyme in complex with statil: an approach to structure-based inhibitor design of the enzyme

Aldose reductase, a monomeric NADPH-dependent oxidoreductase, catalyzes the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The X-ray structure of human aldose reductase holoenzyme in complex with statil was determined at a resolution of 2.1 A. The carboxylate group of statil interacted with the conserved anion binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Statil's hydrophobic phthalazinyl ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group penetrating the "specificity" pocket. The interactions between the inhibitor's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding to residues Leu300 and Thr113. Based on the model of the ternary complex, the program GRID was used in an attempt to design novel potential inhibitors of human aldose reductase with enhanced binding energies of the complex. Molecular modeling calculations suggested that the replacement of the fluorine atom of statil with a carboxylate functional group may enhance the binding energies of the complex by 33%.     

Depolymerisation of F-actin to G-actin and its repolymerisation in the presence of analogs of adenosine triphosphate.

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring. The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect. Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide--8-bromoadenine dinucleotide, nicotinamide--8-thioadenine dinucleotide and nicotinamide 1: N6-ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes. Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding. The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary by not ternary complexes. The case of 1,4,5,6-tetrahydronicotinamide--adenine dinucleotide which does not give ternary complexes like NADH is discussed. The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described. The protective effects of coenzyme analogues towards essential -SH group were also studied.     

X-ray studies of the binding of Cibacron blue F3GA to liver alcohol dehydrogenase

The binding of Cibacron F3GA to orthorhombic crystals of liver alcohol dehydrogenase has been studied to 0.37-nm resolution. Similarities in the binding of this dye were found for rings B, C and D with the binding of the coenzyme NAD+. However, ring A of the dye and the nicotinamide ribose part of the coenzyme are quite differently bound to the enzyme.