Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Short, strong hydrogen bonds at the active site of human acetylcholinesterase: proton NMR studies

Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 +/- 0.02 A, in agreement with the length of 2.63 +/- 0.03 A, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 +/- 0.02 and 2.65 +/- 0.02 A, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.10 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 A, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 A, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 A) and sarin (2.53 +/- 0.26 A) and at low pH (2.52 +/- 0.25 A). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.     

15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism

Nitrogen-15 NMR spectroscopy has been used to study the hydrogen-bonding interactions involving the histidyl residue in the catalytic triad of alpha-lytic protease in the resting enzyme and in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The 15N shifts indicate that a strong hydrogen bond links the active site histidine and serine residues in the resting enzyme in solution. This result is at odds with interpretations of the X-ray diffraction data of alpha-lytic protease and of other serine proteases, which indicate that the serine and histidine residues are too far apart and not properly aligned for the formation of a hydrogen bond. In addition, the nitrogen-15 shifts demonstrate that protonation of the histidine imidazole ring at low pH in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate triggers the disruption of the aspartate-histidine hydrogen bond. These results suggest a catalytic mechanism involving directed movement of the imidazole ring of the active site histidyl residue.     

Regulation of enzymatic activity: one possible role of dietary boron in higher animals and humans

It is well established that vascular plants, diatoms, and some species of marine algal flagellates have acquired an absolute requirement for boron (B), although the primary role remains unknown. Discovery of naturally occurring organoboron compounds, all iono phoric macrodiolide antibiotics with a single B atom critical for activity, established at least one biochemical role of B. The unusual nature of B chemistry suggests the possibility of a variety of biological roles for B. At physiological concentrations and pH, B may react with one N group or one to four hydroxyl groups on specific biological ligands with suitable configuration and charge to form dissociable organoboron compounds or complexes. Suitable ligands include pyridine (e.g., NAD⁺ or NADP) or flavin (e.g., FAD) nucleotides and serine proteases (SP). B reacts with thecis adjacent hydroxyls on the ribosyl moiety of the nucleotides or, in the serine proteases, the N on the imidazole group of histidine or the hydroxyl group on the serine moiety. Reversible inhibition by B of activity of SP or oxidoreductases that require pyridine or flavin nucleotides is well known. Therefore, a proposed essential role for B is as a regulator of relevant pathways, including respiratory burst, that utilize these enzymes. U.S. Department of Agriculture, Agricultural Research Services, Northern Plains Area is an equal opportunity/affirmative action employer, and all agency services are available without discrimination.     

Structure of a histidine ligand in the photosynthetic oxygen-evolving complex as studied by light-induced fourier transform infrared difference spectroscopy.

Fourier transform infrared (FTIR) signals of a histidine side chain were identified in flash-induced S(2)/S(1) difference spectra of the oxygen-evolving complex (OEC) of photosystem II (PS II) using PS II membranes from globally (15)N-labeled spinach and PS II core complexes from Synechocystis cells in which both the imidazole nitrogens of histidine were selectively labeled with (15)N. A negative band at 1113-1114 cm(-1) was downshifted by 7 cm(-1) upon both global (15)N-labeling and selective [(15)N]His labeling, and assigned to the C-N stretching mode of the imidazole ring. This band was unaffected by H-D exchange in the PS II preparations. In addition, several peaks observed at 2500-2850 cm(-1) all downshifted upon global and selective (15)N-labeling. These were ascribed to Fermi resonance peaks on a hydrogen-bonding N-H stretching band of the histidine side chain. FTIR measurements of model compounds of the histidine side chain showed that the C-N stretching band around 1100 cm(-)(1) can be a useful IR marker of the protonation form of the imidazole ring. The band appeared with frequencies in the following order: Npi-protonated (>1100 cm(-1)) > imidazolate > imidazolium > Ntau-protonated (<1095 cm(-1)). The frequency shift upon N-deuteration was occurred in the following order: imidazolium (15-20 cm(-1)) > Ntau-protonated (5-10 cm(-1)) > Npi-protonated approximately imidazolate ( approximately 0 cm(-1)). On the basis of these findings together with the Fermi resonance peaks at >2500 cm(-1) as a marker of N-H hydrogen-bonding, we concluded that the histidine residue in the S(2)/S(1) spectrum is protonated at the Npi site and that this Npi-H is hydrogen bonded. This histidine side chain probably ligated the redox-active Mn ion at the Ntau site, and thus, oxidation of the Mn cluster upon S(2) formation perturbed the histidine vibrations, causing this histidine to appear in the S(2)/S(1) difference spectrum.     

Geometry of interaction of metal ions with histidine residues in protein structures

An analysis of the geometry and the orientation of metal ions bound to histidine residues in proteins is presented. Cations are found to lie in the imidazole plane along the lone pair on the nitrogen atom. Out of the two tautomeric forms of the imidazole ring, the NE2-protonated form is normally preferred. However, when bound to a metal ion the ND1-protonated form is predominant and NE2 is the ligand atom. When the metal coordination is through ND1, steric interactions shift the side chain torsional angle, chi 2 from its preferred value of 90 or 270 degrees. The orientation of histidine residues is usually stabilized through hydrogen bonding; ND1-protonated form of a helical residue can form a hydrogen bond with the carbonyl oxygen atom in the preceding turn of the helix. A considerable number of ligands are found in helices and beta-sheets. A helical residue bound to a heme group is usually found near the C-terminus of the helix. Two ligand groups four residues apart in a helix, or two residues apart in a beta-strand are used in many proteins to bind metal ions.     

Solid-phase active site inhibitors of proteinases.

Phenylalanine chloromethyl ketone covalently attached to porous glass beads was synthesized to serve as a solid-phase active site directed inhibitor of chymotrypsin-like proteolytic enzymes. The solid-phase reagent inhibited 20 nmol of bovine chymotrypsin per gram of glass and covalently bound 30 nmol of protein per gram of glass. Sepharose-bound lysine chloromethyl ketones were synthesized to serve as inhibitors of trypsin-like enzymes. Sepharose-MethionylLysyl chloromethyl ketone inactivated and bound about 6.8 nmol of enzyme per ml of settled gel. In a preliminary experiment, a cyanogen bromide cleavage of the methionine residues showed that it should be possible to release all peptides but the peptide containing the active-site histidine. The immobilized trypsin was also reduced, carboxymethylated and digested with chymotrypsin. The potential of the solid-phase approach is in the isolation of a specific serine proteinase and in the sequence determination of residues surrounding the active-site histidine.     

Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters

The structures of the complexes with alpha-lytic protease of both phosphorus stereoisomers of N-[(2S)-2-[[[(1R)-1-[N-[(tert-butyloxycarbonyl)-L-alanyl-L-alanyl- L-prolyl]amino]-2-methylpropyl]-phenoxyphosphinyl]oxy]propanoyl]- L-alanine methyl ester, an analogue of the peptide Boc-Ala-Ala-Pro-Val-Ala-Ala where Val is replaced with an analogous phosphonate phenyl ester and the subsequent Ala is replaced with lactate, have been determined to high resolution (1.9 A) by X-ray crystallography. Both stereoisomers inactivate the enzyme but differ by a factor of 2 in the second-order rate constant for inactivation [Sampson, N. S., & Bartlett, P. A. (1991) Biochemistry (preceding paper in this issue)]. One isomer (B) forms a tetrahedral adduct in which the phosphonate phenyl ester is displaced by the active site serine (S195) and interacts with the enzyme across seven substrate recognition sites that span both sides of the scissile bond. Seven hydrogen bonds are formed with the enzyme, and 510 A2 of hydrophobic surface area is buried when the inhibitor interacts with the enzyme. Although two hydrogen bonds are gained by incorporation of two residues on the C-terminal side of the scissile bond into the inhibitor, there is very little adjustment in the structure of the enzyme in this region. Surprisingly, the active site histidine (H57) does not interact with the phosphonate, apparently because the phosphonate lacks negative charge in or near the oxyanion hole, and instead, the side chain rotates out of the active site cleft and hydrogen bonds with solvent. The other isomer (A) forms a mixture of two different tetrahedral adducts in the active site, both covalently bonded to Ser 195. One adduct, at approximately 58% occupancy, is exactly the same in structure as the complex formed with isomer B, and the other adduct, at 42% occupancy, has lost the two residues C-terminal to the scissile bond by hydrolysis. In the lower occupancy structure, His 57 does not rotate out of the active site and forms a hydrogen bond with the phosphonate oxygen instead. The structures of both complexes were insensitive to pH. As very little change in structure accompanies the histidine rotation, the complex with isomer B provides an excellent mimic for the structure of the transition state (or high-energy reaction intermediate) that spans both sides of the scissile bond.     

Design and kinetic characterization of multisubstrate inhibitors of dopamine beta-hydroxylase

The synthesis and kinetics characterization of a new class of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) inhibitor, 1-(4-hydroxybenzyl)imidazole-2-thiol, is reported. These inhibitors, which incorporate a phenethylamine substrate mimic and an oxygen mimic into a single molecule, exhibit both the kinetic properties and the potency (Kis approximately 10(-9) M) expected for a multisubstrate inhibitor and are therefore classified as such. Steady-state kinetic experiments with these multisubstrate inhibitors and their substructural analogues support the recently proposed pH-dependent changes in substrate binding order [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3106] and a mechanism whereby the inhibitor binds specifically to the reduced Cu+ form of enzyme at both the phenethylamine substrate site and the active-site copper atom(s). A Yonetani-Theorell double-inhibition experiments indicates mutually exclusive binding of the inhibitor substructures p-cresol and 1-methylimidazole-2-thiol to suggest an extremely short intersite distance between the phenethylamine binding site and the active-site copper atom(s).     

Ab Initio Quantum Mechanics Analysis of Imidazole C-H…O Water Hydrogen Bonding and a Molecular Mechanics Forcefield Correction

Abstract

While it is well established that classical hydrogen bonds play an important role in enzyme structure, function and dynamics, the role of weaker, but ‘activated’ C-H donor hydrogen bonds is poorly understood. The most important such case involves histidine which often plays a direct role in enzyme catalysis and possesses the most acidic C-H donor group of the standard amino acids. In the present study, we obtained optimized geometries and hydrogen bond interaction energies for C-H…O hydrogen bonded complexes between methane, ethylene, benzene, acetylene, and imidazole with water at the MP2-FC/6-31++G(2d,2p) and MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) levels of theory. A strong linear relationship is obtained between the stability of the various hydrogen bonded complexes and both separation distances for H…0 and C—O. In general, these calculations indicate that C-H…0 interactions can be classified as hydrogen bonding interactions, albeit significantly weaker than the classical hydrogen bonds, but significantly stronger than just van der Waals interactions. For instance, while the electronic energy of stabilization at the MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) level of theory of a water C-H…O water hydrogen bond is 4.36 kcal/mol more stable than the methane C-H…O water interaction, the water-water hydrogen bond is only 2.06 kcal/mol more stable than the imidazole C_e?H…O water hydrogen bond. Neglecting this latter hydrogen bonding interaction is obviously unacceptable. We next compare the potential energy surfaces for the imidazole C_e?H…O water and imidazole N_d?H…O hydrogen bonded complexes computed at the MP2/6-31++G(2d,2p) level of theory with the potential energy surface computed using the AMBER molecular mechanics program and forcefields. While the Weiner et al and Cornell et al AMBER forcefields reasonably account for the imidazole N-H…O water interaction, these forcefields do not adequately account for the imidazole C_e?H…O water hydrogen bond. A forcefield modification is offered that results in excellent agreement between the ab initio and molecular mechanics geometry and energy for this C-H…O hydrogen bonded complex.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. II. Polarization of histidine 57 by substrate analogues and competitive inhibitors

The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin A_δ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regions. Borate ion, benzene boronic acid and 2-phenylethylboronic acid, when bound to chymotrypsin at pH 9.5 shift the resonance position of the His-Asp hydrogen bonded proton to ?15.9, ?16.3 and ?17.2 parts per million, respectively. These positions are intermediate between the low pH position in the free enzyme of ?18.0 parts per million and the high pH position of ?14.9 parts per million. The presence of these analogues prevents the His-Asp proton resonance from titrating in the region of pH 6 to 9.5. Similar low field shifts are observed for the hydrogen bonded proton resonance of subtilisin BPN′ when complexed with these boronic acids. The results support the chemical and crystallographic data which show that negatively charged tetrahedral adducts of the boronic acid substrate analogues are formed at the active sites of these enzymes. When combined with similar nuclear magnetic resonance data for the binding of N-acetyl-l-tryptophan to chymotrypsin A_δ, they suggest that a direct interaction occurs between the active site histidine and the atom occupying the leaving group position of the substrate, presumably a hydrogen bond.The His-Asp proton resonance was also monitored in complexes of chymotrypsin A_δ with bovine pancreatic trypsin inhibitor over the pH range 4 to 9. In the complex the low field proton resonance had a field position of ?14.9 parts per million over the pH range 4 to 9 indicating that His57 is in the neutral form, similar to the active enzyme at high pH.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region

The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 Å resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.     

Haem ligands of the ferricytochrome c of Ustilago sphaerogena

The mammalian-type cytochrome c of the basidiomycete Ustilago sphaerogena contains in a single polypeptide chain of 107 residues, two histidine residues located at positions 18 and 33, and one methionine residue situated at position 80 (Bitar et al., 1972). The reaction of Ustilago ferricytochrome c with bromoacetate at neutral pH resulted in the modification of histidine-33, but not of histidine-18 or of the invariant methionine residue. The activities of Ustilago cytochrome c with mitochondrial cytochrome c oxidase and with NADH-cytochrome c reductase were unaltered by the modification. The equilibrium constants for the formation of low-spin complexes of the ferrihaem octapeptide of horse cytochrome c (residues 14-21, including the haem bound covalently to cysteines 14 and 17) with imidazole, N(2)-acetylhistidine and monocarboxymethyl derivatives of N(2)-acetylhistidine were determined spectrophotometrically. Alkylation of the imidazole side-chain group of N(2)-acetylhistidine resulted in a marked decrease in its ability to form low-spin ferrihaem complexes. These results indicate that in Ustilago ferricytochrome c in solution histidine-33 is not involved in the central co-ordination complex. Since side-chain groups of residues other than histidine and methionine do not appear to be involved in the central complexes of other mammalian-type cytochromes c (Hettinger & Harbury, 1964, 1965; Myer & Harbury, 1965) it is likely that in Ustilago ferricytochrome c in solution at neutral pH, the side-chain groups of histidine-18 and methionine-80 are involved in the central co-ordination complex. The latter is stable over the pH range 2.6-8.4.     

Galactose-1-phosphate uridylyltransferase: identification of histidine-164 and histidine-166 as critical residues by site-directed mutagenesis

Galactose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism involving the compulsory formation of a uridylyl enzyme intermediate. The uridylyl group is covalently bonded to the N3 position of a histidine residue in the uridylyl enzyme. The galT gene of Escherichia coli, which codes for the uridylyltransferase and is contained in a plasmid for transformation of E. coli, has been sequenced, and the positions of the 15 histidine residues have been determined from the deduced amino acid sequence of this protein. Fifteen mutant genes, in each of which one of the 15 histidine codons has been changed to an asparagine codon, have been generated and used to transform the E. coli strain JM101. When extracts of the transformants were assayed for uridylyltransferase, 13 exhibited high levels of activity. Two of the extracts containing mutant uridylyltransferase exhibited less than control levels of activity. These mutant proteins, H164N and H166N, were overexpressed, isolated, and tested for their ability to form the compulsory uridylyl enzyme intermediate. Neither the H164N nor the H166N mutant proteins could form the intermediate. Thus, both His-164 and His-166 are critical for activity, and their proximity suggests that both are in the active site. One is the essential nucleophilic catalyst to which the uridylyl group is bonded in the intermediate, and the other serves an equally important, as yet unknown, function. The active-site sequence His(164)-Pro-His(166) is conserved in this enzyme from E. coli, humans, Saccharomyces, and Streptomyces.     

Nitrosyl-heme structures of Bacillus subtilis nitric oxide synthase have implications for understanding substrate oxidation

The crystal structures of nitrosyl-heme complexes of a prokaryotic nitric oxide synthase (NOS) from Bacillus subtilis (bsNOS) reveal changes in active-site hydrogen bonding in the presence of the intermediate N(omega)-hydroxy-l-arginine (NOHA) compared to the substrate l-arginine (l-Arg). Correlating with a Val-to-Ile residue substitution in the bsNOS heme pocket, the Fe(II)-NO complex with both l-Arg and NOHA is more bent than the Fe(II)-NO, l-Arg complex of mammalian eNOS [Li, H., Raman, C. S., Martasek, P., Masters, B. S. S., and Poulos, T. L. (2001) Biochemistry 40, 5399-5406]. Structures of the Fe(III)-NO complex with NOHA show a nearly linear nitrosyl group, and in one subunit, partial nitrosation of bound NOHA. In the Fe(II)-NO complexes, the protonated NOHA N(omega) atom forms a short hydrogen bond with the heme-coordinated NO nitrogen, but active-site water molecules are out of hydrogen bonding range with the distal NO oxygen. In contrast, the l-Arg guanidinium interacts more weakly and equally with both NO atoms, and an active-site water molecule hydrogen bonds to the distal NO oxygen. This difference in hydrogen bonding to the nitrosyl group by the two substrates indicates that interactions provided by NOHA may preferentially stabilize an electrophilic peroxo-heme intermediate in the second step of NOS catalysis.     

Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: function of the Asn-His interaction in the catalytic triad

Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant. Asparagine 156 is not involved in maintaining the overall active-site configuration and does not contribute significantly to the thermal stability of OMPLA. The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain. Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant.     

Hydrogen bond stereochemistry in protein structure and function

Fifty high resolution protein structures from the Brookhaven Protein Data Bank have been analyzed for recurring motifs in hydrogen bond stereochemistry. Although an exhaustive analysis of hydrogen bond statistics has been presented by Baker & Hubbard, a detailed stereochemical analysis of classical donor (N-H, O-H, or S-H) and acceptor (N:, O:, or S:) structure within proteins is lacking. Here, we describe the preferential hydrogen bond stereochemistry for the side-chains of glutamate and aspartate (carboxylate), glutamine and asparagine (carboxamide), arginine (guanidinium), histidine (imidazole/imidazolium), tryptophan (indole), tyrosine (phenolic hydroxyl), lysine (ammonium), serine and threonine (alkyl hydroxyl), cysteine (thiol), methionine (thioether) and cystine (disulfide). Preferential hydrogen bond stereochemistry is governed by (1) the electronic configuration of acceptor atoms, (2) the steric accessibility of donor atoms and (3) the conformation of amino acid side-chains. Applications of hydrogen bond stereochemistry are useful in the interpretation of protein structure, function and stability. Additionally, this stereochemistry is a prerequisite to the interpretation of protein-other molecule recognition and biological catalysis.     

15N and 1H NMR studies of Rhodospirillum rubrum cytochrome c2

15N-Enriched cytochrome c2 was purified from Rhodospirillum rubrum that had been grown on 15NH4Cl, and the diamagnetic iron(II) form of the cytochrome was studied by 15N and 1H NMR spectroscopy. 15N resonances of the four pyrrole nitrogens, the ligand histidine nitrogens, the highly conserved tryptophan indole nitrogen, and some proline nitrogens are assigned. The resonances of the single nonligand histidine are observed only at low pH because of severe broadening produced by proton tautomerization. The resonances of exchangeable protons bonded to the nitrogens of the ligand histidine, the tryptophan, and some amide groups are also assigned. The exchange rates of the nitrogen-bound protons vary greatly: most have half-lives of less than minutes, the indolic NH of Trp-62 exchanges with a half-time of weeks, and the ligand histidine NH proton exchanges with a half-time of months. The latter observation is indicative of extreme exclusion of solvent from the area surrounding the ligand histidine and lends credence to theories implicating the degree of hydrophobicity in this region as an important factor in adjusting the midpoint potential. The dependence of the 15N and 1H NMR spectra of ferrocytochrome c2 on pH indicates neither the Trp-62 nor the ligand His side chains become deprotonated to any appreciable extent below pH 9.5. The His-18 NH remains hydrogen bonded, presumably to the Pro-19 carboxyl group, throughout the pH titrations. Because neither deprotonated nor non-hydrogen-bonded forms of His-18 are observed in spectra of the ferrocytochrome, the participation of such forms in producing a heterogeneous population having different g tensor values seems unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. 15N NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pKa of histidine-95 is below 4.5. This abnormally low pKa rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. 15N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N epsilon 2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)