Refined X-ray structure of the low pH form of ribonuclease T1-2'-guanylic acid complex at 1.9 A resolution

The three-dimensional X-ray structure of the RNase T1[EC 3.1.27.3]-2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 A resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His27 and His40 interact with the carboxyl side chains of Glu82 and Glu58, respectively, whereas that of His92 is in contact with the main chain carbonyl oxygen of Ala75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche(+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and O5', and the 2'-phosphate orientation is trans-gauche(-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1--Glu46O epsilon 2, N2---Asn98O,O6---Asn44N, and N7 ---Asn43N delta 2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77N eta 2, Glu58O episilon 2, and Tyr 38O eta but not with any of the histidine residues. Arg77N eta 2 also interacts with Tyr38O eta. There is no interaction between the ribose moiety of the inhibitor and the enzyme.     

The structures of RNase A complexed with 3'-CMP and d(CpA): active site conformation and conserved water molecules.

The interactions of RNase A with cytidine 3'-monophosphate (3'-CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.     

Heavy metal-pyrimidine nucleotide interaction: x-ray structure of a cadmium derivative of cytidine 5''-monophosphate.

An X-ray crystal-structure determination has shown that the compound [Cd(5'-CMP)(H2O)],H2O has a polymeric structure in which each cadmium atom is bonded to five atoms: to the N(3) position on the base, to a phosphate oxygen from each of three other 5'-CMP groups and to a water molecule.     

Neutron diffraction study of carbonmonoxymyoglobin.

Neutron diffraction data from a crystal of carbonmonoxymyoglobin were refined by PROLSQ, a modern restrained least-squares procedure in reciprocal space, in conjunction with a solvent analysis technique, to a final R-factor of 11.3%. The ligand CO occupies two sites and its binding conformations are distorted from the linear conformation. The N epsilon atom of the distal histidine residue is deprotonated (not deuterated), and a water molecule is bound to the N delta atom of the distal histidine. The side-chain of Lys56 (D6) exists in two alternative charge-binding sites. His24 (B5) and His119 (GH1) share a hydrogen atom. His12 (A10) and His36 (C1) are deprotonated. The deprotonated imidazole ring of His12 (A10) may act as a hydrogen-bond acceptor. The heme group is planar within 0.09 A root-mean-square (r.m.s.) deviation from planarity. The solvent environments for the two propionic acid groups are different. The side-chain of Arg45 (CD3) forms hydrogen bonds with the side-chain of Asp60 (E3) and one of the two propionic acid groups. An average N-2H . . . O angle in helical regions is 147 (+/- 11) degrees. Eleven main-chain amide hydrogen atoms from hydrophobic residues do not exchange with deuterium. The overall atomic occupancy factors for the main-chain and side-chain atoms are quite uniform, at 0.97 (+/- 0.07) and 0.93 (+/- 0.10), respectively, as shown by an occupancy analysis made at the end of the refinement procedure.     

The specific guanine binding site of the ribonuclease T1 family enzymes and of G-proteins is modeled in the cocrystal formed by 7-methylguanosine-5'-phosphate and phenylalanine

In the cocrystal formed by 7-methylguanosine-5'-phosphate.phenylalanine.6H2O, the interactions between guanine and phenylalanine are similar to those observed in the complex of ribonuclease T1 with 2'-guanylic acids, and those of the two G-proteins, Elongation Factor-Tu and ras oncogene p21, with GDP. They are similar in the following three points: (a) guanine N(1)H and N(2)H donate cyclic N-H...O hydrogen bonds to the carboxylate group of phenylalanine in the former cocrystal and to the side chain carboxylate group of Asp or Glu in the latter proteins, (b) O(6) of guanine accepts hydrogen bond(s) from main-chain NH group(s), and (c) the purine moiety is sandwiched between aromatic (or hydrophobic) amino acid side chains.     

Assignment of the imidazole ring nitrogen protons of histidine 48 in the proton NMR spectrum of ribonuclease A in water solution.

Several exchangeable resonances, designated a, b, c and d are observed in the 11-14 ppm (from 2,2-dimethyl-2-silapentane-5-sulfonate) region of the proton spectrum of ribonuclease A in water solution. We describe a number of lines of evidence suggesting the assignment of peaks b and c to the N1 and N3 protons of His 48, which occupies an interior position in the protein remote from the active site. This evidence includes the observation that the binding of Cu(II) and 3'-CMP (cytidine 3'-monophosphate) has no effect on these resonances. Further evidence includes pH titration data showing a pKa of approx. 2 for these protons, solvent exchange rates in the native state and with disulfide bridges IV-V and III-VIII cleaved, the observation of the carboxymethylated enzymes CM-His12-RNAase A and CM-His119-RNAase A, and of the modified enzymes Des(1-21)-RNAase A (S-protein) and Des(119-124)-RNAase A.     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.     

Structure of azurin from Alcaligenes denitrificans refinement at 1.8 A resolution and comparison of the two crystallographically independent molecules

The structure of the blue copper protein azurin, from Alcaligenes denitrificans, has been refined crystallographically by restrained least-squares methods. The final crystallographic R value for 21,980 observed reflections to 1.8 A (1 A = 0.1 nm) resolution is 0.157. The asymmetric unit of the crystal contains two independent azurin molecules, the model for which comprises 1973 protein atoms, together with three SO2-4 ions, and 281 water molecules. Comparison of the two molecules shows very high correspondence. For 125 out of 129 residues (excluding only the chain termini, residues 1 to 2 and 128 to 129) the root-mean-square (r.m.s.) deviation in main-chain atom positions is 0.27 A. For other structural parameters r.m.s. deviations are also low; torsion angles 6.5 degrees, hydrogen bond lengths 0.12 A, bonds to copper 0.04 A and bond angles at the copper 3.9 degrees. The only significant differences are at the chain termini and in several loops. Some of these can be attributed to crystal packing effects, others to genuine structural microheterogeneity. Refinement has confirmed that the copper co-ordination is best described as distorted trigonal planar, with strong in-plane bonds to His46 N delta 1, His117 N delta 1 and Cys112 S gamma, and much weaker axial interactions with Met121 S delta and Gly45 C = O. Two N-H...S hydrogen bonds characterize Cys112 S gamma as a thiolate (S-) sulphur and may influence the visible absorption maximum. Atoms in and around the copper site have very low mobility, whereas the most mobile regions of the molecule are the chain termini and some of the connecting loops between secondary structure elements, especially those at the "southern" end, remote from the copper site. Main-chain to side-chain hydrogen bonds supply important stabilizing interactions at the "northern" end. Surface features include the hydrophobic patch around His117, probably important for electron transfer, the SO2-4 site at His83, and the general absence of ion pairs, despite the presence of many charged amino acid residues. The 281 water molecules include 182 that occur as approximately twofold-related pairs. There are no internal water molecules. The water sites common to both azurin molecules include those in surface pockets and some in intermolecular contact regions. They are characterized by relatively low thermal parameters and numerous protein contacts.     

Carbon-13 NMR investigations on ribonuclease A.

The proposed interaction between the amino acid residues Asp 14 and His 48 of ribonuclease A has been confirmed by 13C-NMR spectroscopy. The titration behaviour of the resonance of the side-chain carboxyl group of Asp 14 suggests a pKa of 6.5--7.0 for His 48. An equilibrium between different conformation process of His 48. Upon this deprotonation a hydrogen bond between the side-chains of Asp 14 or His 48 and Tyr 25 seems to be formed as is suggested by the behaviour of a tyrosine C zeta resonance assigned to Tyr 25. One phenylalanine resonance broadens and moves upfield on the addition of the inhibitor Cyd-2'-P, being therefore assigned to Phe 120. The behaviour of this resonance suggests that the upfield shift results from the anisotropy of the cytidine ring. Three signals are assigned to the three Phe residues.     

Enzyme specificity: base recognition and hydrolysis of RNA by ribonuclease A

The substrate specificity of pancreatic ribonuclease A is discussed in light of observations based on accurate X-ray structure analysis of several enzyme-nucleotide complexes. A hypothesis for protein-nucleic acid recognition is presented which proposes that: (a) pyrimidine bases in RNA are recognised by ribonuclease due to the charge complementarity of two groups (the amide nitrogen and the side chain oxygen (OG) of threonine 45) of the protein and relevant atoms in the heterocyclic base (O2 and N3 in pyrimidine nucleotides); (b) interaction of the protein with the ribose moiety of the nucleotides is non-specific; and (c) conformational flexibility in the region of the scissile P-O bond is provided by different locations of the phosphoryl oxygens, rather than by an overall translation of the phosphate moiety.     

Specificity of pancreatic ribonuclease-A. An X-ray study of a protein-nucleotide complex

The modified purine nucleotide 8-oxo-guanosine-2'-phosphate binds at the pyrimidine binding site of ribonuclease-A. The O8-2'GMP inhibitor is in a syn conformation, with an intramolecular hydrogen bond between the N-3 atom of the base and the O-5' atom of the ribose. The essential groups of the protein involved in base recognition are O gamma 45 and N-45, which form hydrogen bonds to the five-membered ring of the heterocyclic base. Mobility of enzyme side-chains (viz. Lys41, Lys66, His119) close to the catalytic cleft of the protein allows conformational flexibility in the substrate binding region of ribonuclease-A. Inhibitor binding alters the solvent structure of the protein but the overall shape of the enzyme is not effected.     

Further evidence for an allosteric model for ribonuclease.

Evidence is presented from three experimental systems to support the allosteric model of Walker et al. (1975) (Biochem. J. 147, 425-433) which explains the substrate-concentration-dependent transition observed in the RNAase (ribonuclease)-catalysed hydrolysis of 2':3'-cyclic CMP (cytidine 2':3'-cyclic monophosphate). 1. Kinetic studies of the initial rate of hydrolysis of 2':3'-cyclic CMP show that the midpoint of the transition shifts to lower concentrations of 2':3'-cyclic CMP in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, 3'-UMP and Pi; 2'-CMP and 2'-UMP do not cause such a shift. 2. Trypsin-digestion studies show that a conformational change in RNAase to a form less susceptible to tryptic inactivation is induced in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, and 3'-UMP. 2'-CMP, 2'-AMP and 2'-UMP do not induce this conformational change. 3. Equilibrium-dialysis experiments demonstrate the multiple binding of molecules of 3'-CMP, 3'-AMP and 5'-AMP to a molecule of RNAase. 2'-CMP binds the ratio 1:1 over the analogue concentration range studied.     

1H and 15N NMR investigation of the interaction of pyrimidine nucleotides with ribonuclease A

Extensive 1H and 15H NMR investigations of the nucleotide moieties capable of hydrogen bonding to ribonuclease A were carried out in order to gain more detailed information on the specificity of nucleotide-enzyme interaction. The 1H investigations focussed on those protons presumed to be involved in hydrogen bonding between the various nucleotides and the enzyme. In particular these were the imino protons of the uridine nucleotides and the amino protons of the cytidine nucleotides. The technique of 15N-1H double quantum filtering was applied for observation of the resonances of the latter in the nucleotide-enzyme complex. The downfield shift observed for the imino proton resonance of the uridine nucleotides was indicative of hydrogen bond formation to the enzyme. 15N NMR spectra of the free nucleotides and the nucleotide-enzyme complexes were also acquired to examine the possibility of hydrogen bond formation at the N3 site of both pyrimidine bases and the amino group of the cytidine nucleotides. The downfield shift observed for the 15N3 resonance of the uridine nucleotides and the upfield shift observed for the corresponding resonance of the cytidine nucleotides was evidence that the N3 moiety acts as hydrogen donor or hydrogen acceptor in the nucleotide-enzyme complex. The effect of complex formation on the 15N1 resonance of the respective bases was also studied. Both 1H and 15N NMR results indicated subtle differences between the complexes of the 2' and 3' nucleotides. The extent of hydrogen bonding as well as the arrangement of the nucleotide base at the active site of the enzyme varies in dependence on the position of the phosphate group. It is established that hydrogen bonding, though not the main binding force between the nucleotides and the enzyme, is certainly a major factor of RNase A specificity for pyrimidine nucleotides.     

Conformational stability of ribonuclease A complexes with specific inhibitors

Isotermic unfolding of ribonuclease A, phosphopyridoxyl-8Lys41-RNAase A and complexes of the enzyme with cytidine, 2'-CMP, 3'-CMP, 3'-AMP and with the phosphoric ester of 1-(omega-oxypropyl)-cytosine in presence of urea has been studied. The stabilization of the protein structure resulting from the complex formation was shown to be determined by the ligand nucleobase binding. The comparison of the results obtained with those known from the literature suggests, that binding and catalytic zones of the enzyme active site form an integrated network system which is substained by multipoint contacts between the constituents. The change in the state of any part within the enzyme active state affects the energetics of the whole protein globule.     

The occurrence of C--H...O hydrogen bonds in alpha-helices and helix termini in globular proteins

A comprehensive database analysis of C--H...O hydrogen bonds in 3124 alpha-helices and their corresponding helix termini has been carried out from a nonredundant data set of high-resolution globular protein structures resolved at better than 2.0 A in order to investigate their role in the helix, the important protein secondary structural element. The possible occurrence of 5 --> 1 C--H...O hydrogen bond between the ith residue CH group and (i - 4)th residue C==O with C...O < or = 3.8 A is studied, considering as potential donors the main-chain Calpha and the side-chain carbon atoms Cbeta, Cgamma, Cdelta and Cepsilon. Similar analysis has been carried out for 4 --> 1 C--H...O hydrogen bonds, since the C--H...O hydrogen bonds found in helices are predominantly of type 5 --> 1 or 4 --> 1. A total of 17,367 (9310 of type 5 --> 1 and 8057 of type 4 --> 1) C--H...O hydrogen bonds are found to satisfy the selected criteria. The average stereochemical parameters for the data set suggest that the observed C--H...O hydrogen bonds are attractive interactions. Our analysis reveals that the Cgamma and Cbeta hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic beta-branched residue Ile to participate in 5 --> 1 C--H...O hydrogen bonds involving methylene Cgamma 1 atom as donor in alpha-helices. This may be an enthalpic compensation for the greater loss of side-chain conformational entropy for beta-branched amino acids due to the constraint on side-chain torsion angle, namely, chi1, when they occur in helices. The preference of amino acids for 4 --> 1 C--H...O hydrogen bonds is found to be more for Asp, Cys, and for aromatic residues Trp, Phe, and His. Interestingly, overall propensity for C--H...O hydrogen bonds shows that a majority of the helix favoring residues such as Met, Glu, Arg, Lys, Leu, and Gln, which also have large side-chains, prefer to be involved in such types of weak attractive interactions in helices. The amino acid side-chains that participate in C--H...O interactions are found to shield the acceptor carbonyl oxygen atom from the solvent. In addition, C--H...O hydrogen bonds are present along with helix stabilizing salt bridges. A novel helix terminating interaction motif, X-Gly with Gly at C(cap) position having 5 --> 1 Calpha--H...O, and a chain reversal structural motif having 1 --> 5 Calpha-H...O have been identified and discussed. Our analysis highlights that a multitude of local C--H...O hydrogen bonds formed by a variety of amino acid side-chains and Calpha hydrogen atoms occur in helices and more so at the helix termini. It may be surmised that the main-chain Calpha and the side-chain CH that participate in C--H...O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N--H...O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins.     

Crystal structure of an RNA quadruplex containing inosine tetrad: implications for the roles of NH2 group in purine tetrads

Polyinosinic acid has been known to adopt the four-stranded helical structure but its basic unit, inosine tetrad (I tetrad), has not been determined at the atomic level. Here we report the crystal structure of an RNA quadruplex containing an I tetrad at 1.4 A resolution. The I tetrad has one cyclic hydrogen bond N1...O6 with the bond length of 2.7 A. A water bridge is observed in the minor groove side of the base tetrad. Even though it is sandwiched by guanine tetrads (G tetrads), the I tetrad is buckled towards the 3' side of the tetrad plane, which results from the different interaction strength with K ions on two sides of the tetrad plane. Comparison with both G tetrad and adenine tetrad indicates that lack of NH2 in the C2 position makes the I tetrad prone to buckle for interactions with ligands. Two U*(G-G-G-G) base pentads are observed at the junction of the 5' termini of two quadruplexes. The uridine residue in the base pentad is engaged in two hydrogen bonding interactions (N2(G)-H...O2(U) and O2'(G)-H...O4(U)) and a water-mediated interaction (N3(G) and N3(U)) with the G tetrad. We also discuss the roles of amino group in purine tetrads and the inter-quadruplex interactions in RNA molecules. These quadruplexes may interact with each other by stacking, groove binding and intercalation.     

15N kinetic isotope effects on uncatalyzed and enzymatic deamination of cytidine.

15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.     

Crystal structures of aspartate carbamoyltransferase ligated with phosphonoacetamide, malonate, and CTP or ATP at 2.8-A resolution and neutral pH

The R-state structures of the ATP and CTP complexes of aspartate carbamoyltransferase ligated with phosphonoacetamide and malonate have been determined at 2.8-A resolution and neutral pH. These structures were solved by the method of molecular replacement and were refined to crystallographic residuals between 0.167 and 0.182. The triphosphate, the ribose, and the purine and pyrimidine moieties of ATP and CTP interact with similar regions of the allosteric domain of the regulatory dimer. ATP and CTP relatively increase and decrease the size of the allosteric site in the vicinity of the base, respectively. For both CTP and ATP at pH 7, the gamma-phosphates are bound to His20 and are also near Lys94, while the alpha-phosphates interact exclusively with Lys94. The 2'-hydroxyls of both CTP and ATP are near the amino group of Lys60. The pyrimidine ring of CTP makes specific hydrogen bonds at the allosteric site: the NH2 group donates hydrogen bonds to the main-chain carbonyls of Ile12 and Tyr89 and the pyrimidine ring carbonyl oxygen accepts a hydrogen bond from the amino group of Lys60; the nitrogen at position 3 in the pyrimidine ring is hydrogen bonded to a main-chain NH group of Ile12. The purine ring of ATP also makes numerous interactions with residues at the allosteric site: the purine NH2 (analogous to the amino group of CTP) donates a hydrogen bond to the main-chain carbonyl oxygen of Ile12, the N3 nitrogen interacts with the amino group of Lys60, and the N1 nitrogen hydrogen bonds to the NH group of Ile12. The binding of CTP and ATP to the allosteric site in the presence of phosphonoacetamide and malonate does not dramatically alter the structure of the allosteric binding site or of the allosteric domain. Nonetheless, in the CTP-ligated structure, the average separation between the catalytic trimers decreases by approximately 0.5 A, indicating a small shift of the quaternary structure toward the T state. In the CTP- and ATP-ligated R-state structures, the binding and occupancy of phosphonoacetamide and malonate are similar and the structures of the active sites are similar at the current resolution of 2.8 A.     

Using proton nuclear magnetic resonance to study the mode of ribonuclease A inhibition by competitive and noncompetitive inhibitors

The C(2) proton resonances of the active site histidines (His 12 and His 119) of ribonuclease A have been exploited to study the inhibition pattern of both noncompetitive (four green tea polyphenols and their copper complexes) and competitive (3'-O-carboxy esters of thymidine and 3'-amino derivatives of uridine) inhibitors. Competitive inhibitors devoid of any phosphate group have the ability to change the pK(a) of the histidine residues at the active site. Their mode of inhibition, albeit competitive, is found to be different compared to known phosphate inhibitors 2'-CMP and 3'-CMP as revealed by changes in the pK(a) values. We find a correlation between the changes in the chemical shift of His 12 and the corresponding inhibition constants (K(i)).     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.