Role Of His Research Articles

A role for His 155 in binding of human prion peptide 144–167 to immobilised prion protein

The interactions and conformational changes that lead to the conversion of the normal prion protein (PrP c) to its pathogenic form, PrP sc, are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP 144–167) corresponding to residues comprising the α helix 1–β strand 2 domain of PrP c is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His 155 in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP 144–167 revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP 144–167.

Molecular insights into nitrogenase FeMoco insertion – The role of His 274 and His 451 of MoFe protein α subunit

The final step of FeMo cofactor (FeMoco) assembly involves the insertion of FeMoco into its binding site in the molybdenum–iron (MoFe) protein of nitrogenase. Here we examine the role of His α274 and His α451 of Azotobacter vinelandii MoFe protein in this process. Our results from combined metal, activity, EPR, stability and insertion analyses show that mutations of His α274 and/or His α451, two of the histidines that belong to a so-called His triad, to small uncharged Ala specifically reduce the accumulation of FeMoco in MoFe protein. This observation indicates that the enrichment of histidines at the His triad is important for FeMoco insertion and that the His triad potentially serves as an intermediate docking point for FeMoco through transitory ligand coordination and/or electrostatic interaction.

Molecular insights into nitrogenase FeMo cofactor insertion: the role of His 362 of the MoFe protein α subunit in FeMo cofactor incorporation

The assembly of the complex iron-molybdenum cofactor (FeMoco) of nitrogenase molybdenum-iron (MoFe) protein has served as one of the central topics in the field of bioinorganic chemistry for decades. Here we examine the role of a MoFe protein residue (His alpha362) in FeMoco insertion, the final step of FeMoco biosynthesis where FeMoco is incorporated into its binding site in the MoFe protein. Our data from combined metal, activity and electron paramagnetic resonance analyses show that mutations of His alpha362 to small uncharged Ala or negatively charged Asp result in significantly reduced FeMoco accumulation in MoFe protein, indicating that His alpha362 plays a key role in the process of FeMoco insertion. Given the strategic location of His alpha362 at the entry point of the FeMoco insertion funnel, this residue may serve as one of the initial docking points for FeMoco insertion through transient ligand coordination and/or electrostatic interaction.

The structure of GDP‐4‐keto‐6‐deoxy‐d‐mannose‐3‐dehydratase: A unique coenzyme B6‐dependent enzyme

L-colitose is a 3,6-dideoxysugar found in the O-antigens of some Gram-negative bacteria such as Escherichia coli and in marine bacteria such as Pseudoalteromonas tetraodonis. The focus of this investigation, GDP-4-keto-6-deoxy-D-mannose-3-dehydratase, catalyzes the third step in colitose production, which is the removal of the hydroxyl group at C3' of GDP-4-keto-6-deoxymannose. It is an especially intriguing PLP-dependent enzyme in that it acts as both a transaminase and a dehydratase. Here we present the first X-ray structure of this enzyme isolated from E. coli Strain 5a, type O55:H7. The two subunits of the protein form a tight dimer with a buried surface area of approximately 5000 A2. This is a characteristic feature of the aspartate aminotransferase superfamily. Although the PLP-binding pocket is formed primarily by one subunit, there is a loop, delineated by Phe 240 to Glu 253 in the second subunit, that completes the active site architecture. The hydrated form of PLP was observed in one of the enzyme/cofactor complexes described here. Amino acid residues involved in anchoring the cofactor to the protein include Gly 56, Ser 57, Asp 159, Glu 162, and Ser 183 from one subunit and Asn 248 from the second monomer. In the second enzyme/cofactor complex reported, a glutamate ketimine intermediate was found trapped in the active site. Taken together, these two structures, along with previously reported biochemical data, support the role of His 188 as the active site base required for catalysis.

A Conserved Histidine in Insulin Is Required for the Foldability of Human Proinsulin: STRUCTURE AND FUNCTION OF AN ALAB5 ANALOG

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His(B10) is well defined: although not required in the mature hormone for receptor binding, in the islet beta cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His(B5) is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala(B5)-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala(B5) analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala(B5) and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His(B5) in native insulin. Substantial receptor binding activity is retained (80 +/- 10% relative to the parent monomer). Although the thermodynamic stability of the Ala(B5) analog is decreased (DeltaDeltaG(u) = 1.7 +/- 0.1 kcal/mol), consistent with loss of His(B5)-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His(B5) facilitate alignment of Cys(A7) and Cys(B7) in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.

Is histology (His) relevant in adjuvant (Adj)/neoadjuvant (NA) therapy of breast cancer (BC)?

10541 Background: Invasive lobular carcinoma (ILC) constitutes 10–15% of cases of invasive BC. In ongoing NCI-sponsored, randomized trials (RT) of early BC, His is not a stratification factor, despite the suggestion that ILC might have distinct clinical behavior and gene expression profile. Compared with invasive ductal carcinoma (IDC), ILC is more frequently of lower grade and positive for estrogen receptor (ER). Methods: We reviewed the literature to evaluate the effect of His on outcome of Adj and NA therapy, by quantifying its impact on the pathologic response (pCR) rate to NA chemotherapy (CT), and on the results of adjuvant trials. We also compiled retrospective series analyzing pCR to NA CT according to His. Results: We retrieved 16 RT (8 phase III, 8 phase II) of NA therapy; 11 compared CT regimens, 3 regimens that contained CT and hormone therapy (HT), and 2 HT only. None of the RT used His for stratification; 5 studies described His at randomization, but only 1 provided information on pCR rate to CT according to His (Dieras et al, 4.0% for ILC, 8.3% for IDC). Compiled results of 6 retrospective series which included nearly 3,000 patients (PTS) showed a pCR rate of 1.7% (6 of 354) in ILC PTS and of 11.6% (300 of 2584) in IDC PTS (P < 0.01). Of 39 phase III RT of Adj CT, 7 compared CT vs HT, 14 CT vs CT + HT, 11 HT vs HT + CT, and 7 CT vs HT vs CT + HT. Of the 39 RT, only 4 described His in demography (9%, 9%, 10% and 12% of PTS participating in these 4 RT were ILC), but none analyzed results according to His. Conclusions: We suggest that the role of CT in early ILC is not clear. The pCR rate is significantly lower in ILC PTS treated with NA CT. It is also not clear if His is an independent predictive factor or a marker of grade/ER expression. The specific value of CT in the Adj treatment of ILC PTS has not been adressed in the Adj RT. Past and current, ongoing RT of Adj CT do not stratify PTS according to His neither evaluate the results according to His. Given the potential therapeutic implications, we suggest that the role of His should be more thoroughly investigated in future RT in order to define the appropriate effectiveness of CT and HT in the treatment of ILC PTS, so that possible overtreatment of ILC PTS with CT might be avoided. No significant financial relationships to disclose.

Catalytic mechanism of S-adenosylhomocysteine hydrolase: Roles of His 54, Asp130, Glu155, Lys185, and Aspl89

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 Å resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670–22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5′–S D bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3′-oxidation and the C4′-proton abstraction are a concerted event and the C5′–S D bond cleavage is an independent event. The C4′-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3′-keto-4′, 5′-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3′-oxidation, D130 abstracts the C4′-proton, D189, and E155 act as a communicator between the concerted C3′-oxidation and C4′-proton abstraction, and H54 plays as a general acid to cleave the C5′–S D bond of AdoHcy.

Histidine nutrition and genotype affect cataract development in Atlantic salmon, Salmo salar L.

The aim of this study was to investigate effects of dietary levels of histidine (His) and iron (Fe) on cataract development in two strains of Atlantic salmon monitored through parr-smolt transformation. Three experimental diets were fed: (i) a control diet (CD) with 110 mg kg(-1) Fe and 11.7 g kg(-1) His; (ii) CD supplemented with crystalline His to a level of 18 g kg(-1) (HD); and (iii) HD with added iron up to 220 mg kg(-1) (HID). A cross-over design, with two feeding periods was used. A 6-week freshwater (FW) period was followed by a 20-week period, of which the first three were in FW and the following 17 weeks in sea water (SW). Fish were sampled for weighing, cataract assessment and tissue analysis at five time points. Cataracts developed in all groups in SW, but scores were lower in those fed high His diets (P < 0.05). This effect was most pronounced when HD or HID was given in SW, but was also observed when these diets were given in FW only. Histidine supplementation had a positive effect on growth performance and feed conversion ratio (P < 0.05), whereas this did not occur when iron was added. Groups fed HD or HID had higher lens levels of His and N-acetyl histidine (NAH), the latter showing a marked increase post-smoltification (P < 0.05). The HD or HID groups also showed higher muscle concentrations of the His dipeptide anserine (P < 0.05). There was a strong genetic influence on cataract development in the CD groups (P < 0.001), not associated with tissue levels of His or NAH. The role of His and His-related compounds in cataractogenesis is discussed in relation to tissue buffering, osmoregulation and antioxidation.

The conserved histidine 106 of large thioredoxin reductases is likely to have a structural role but not a base catalyst function

The catalytic activity of selenocysteine-containing thioredoxin reductases can be mimicked by cysteine-variants if the local environment at the C-terminal redox center supports thiol activation. This concept of a linear catalytic site was challenged by structural data suggesting that the invariant residue His 106 functions as a base catalyst for the dithiol-disulphide exchange reaction between enzyme and substrate. As reported here, we changed His 106 to asparagine, glutamine, and phenylalanine in various C-terminal mutants of Drosophila melanogaster thioredoxin reductase. The catalytic activity dropped considerably, yet pH-profiles did not reveal differences, rendering a function for His 106 as a base catalyst unlikely. Interestingly, the phenylalanine-mutants, designed as negative controls were the most active mutants which suggests rather a structural role of His 106.

Characterization of the Soluble Domain of the ABC7 Type Transporter Atm1

Atm1 is an ABC transporter that is located in yeast mitochondria and has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins. The soluble nucleotide binding domain of Atm1 (Atm1-C) has been overexpressed in Escherichia coli, purified, and characterized. Dissociation constants (KD) for Atm1-C binding of ATP (KD approximately 97 microm, pH 7.3, and approximately 102 microm, pH 10.0) and ADP (KD approximately 43 microm, pH 7.3, and 92 microm, pH 10.0) were measured by fluorimetry. The higher binding affinity for ADP suggests that the transmembrane-spanning domain may be required to promote a structural change in the nucleotide binding domain to facilitate substrate export and ADP release. ADP also had an inhibitory effect on Atm1-C with an IC50 of 10 mm. The Michaelis-Menten constants Vmax, KM, and kcat of Atm1-C were measured as 1.822 microm min(-1), 513 microm, and 0.055 min(-1), respectively. The metal dependence of Atm1-C ATPase demonstrated a reactivity order of Mn2+ > Mg2+ > Co2+, while Mg2+ and Co2+ were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (napp = 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter.

Membrane Fusion Induced by Vesicular Stomatitis Virus Depends on Histidine Protonation

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. VSV-induced membrane fusion occurs at a very narrow pH range, between 6.2 and 5.8, suggesting that His protonation is required for this process. To investigate the role of His in VSV fusion, we chemically modified these residues using diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited membrane fusion mediated by VSV in a concentration-dependent manner and that the complete inhibition of fusion was fully reversed by incubation of modified virus with hydroxylamine. Fluorescence measurements showed that VSV modification with DEPC abolished pH-induced conformational changes in G protein, suggesting that His protonation drives G protein interaction with the target membrane at acidic pH. Mass spectrometry analysis of tryptic fragments of modified G protein allowed the identification of the putative active His residues. Using synthetic peptides, we showed that the modification of His-148 and His-149 by DEPC, as well as the substitution of these residues by Ala, completely inhibited peptide-induced fusion, suggesting the direct participation of these His in VSV fusion.

Oxidative and hydrolytic properties of beta-amyloid.

beta-Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733-738]. In the present work we compare the ability of beta-amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacetate (DCF-DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of beta-amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human beta-amyloid (1-40), containing three amino-acid substitutions (Arg5-->Gly, Tyr10-->Phe and His13-->Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that beta-amyloid-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H2DCF oxidation did not change or increase DCF-DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox-active metal ions and was practically oxygen independent while the oxidation process was redox-active-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide-mediated singlet oxygen intermediate in this Fenton mechanism-driven reaction. These results indicate that hydrolytic and oxidative properties of beta-amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to beta-amyloid toxicity.

The Role of Histidine 632 in Catalysis by Human Topoisomerase I

Based on the crystal structure of human topoisomerase I, we hypothesized that hydrogen bonding between the side chain of the highly conserved His(632) and one of the nonbridging oxygens of the scissile phosphate contributes to catalysis by stabilizing the transition state. This hypothesis has been tested by examining the effects of changing His(632) to glutamine, asparagine, alanine, and tryptophan. The change to glutamine reduced both the relaxation activity and single-turnover cleavage activity by approximately 100-fold, whereas the same change at three other conserved histidines (positions 222, 367, and 406) had no significant effect on the relaxation activity. The properties of the mutant protein containing asparagine instead of histidine at position 632 were similar to those of the glutamine mutant, whereas mutations to alanine or tryptophan reduced the activity by approximately 4 orders of magnitude. The reduction in activity for the mutants was not due to alterations in substrate binding affinities or changes in the cleavage specificities of the proteins. The above results for the glutamine mutation in conjunction with the similar effects of pH on the wild type and the H632Q mutant enzyme rule out the possibility that His(632) acts as a general acid to protonate the leaving 5'-oxygen during the cleavage reaction. Taken together, these data strongly support the hypothesis that the only role for His(632) is to stabilize the pentavalent transition state through hydrogen bonding to one of the nonbridging oxygens.

Engineering His(E7) Affects the Control of Heme Reactivity in Aplysia limacina Myoglobin

Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 Å crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.

Photodynamic cross-linking of proteins: IV. Nature of the His–His bond(s) formed in the rose bengal-photosensitized cross-linking of N-benzoyl- L-histidine

The photodynamic (photosensitized) cross-linking of N-benzoyl- L-histidine (Bz-His) as a model system was examined as part of a continuing study of the role of His–His intermolecular cross-links in the photosensitized cross-linking of proteins. The illumination of Bz-His in the presence of rose bengal (RB) bound to water insoluble plastic beads in 0.1 M sodium phosphate buffer of pH 7.4 resulted in the covalent cross-linking of the His derivative. The main dimeric cross-linked product ( 1) was isolated using a preparative silica gel 60 column and purified by preparative reverse phase HPLC. The chemical structure of the cross-link was determined using MS, 2D NMR spectral methods and other standard techniques. Product 1 was found to be a dimer of two His residues between the δ2-carbon of one residue (photo-oxidized to the carbonyl functionality at the ε1-carbon) and the ε2-nitrogen of the other residue. The formation of His–His cross-links was mediated by singlet oxygen, as would be expected with RB as the sensitizer. A mechanism for the formation of the cross-link was proposed in which the first step was the 1,4-cycloaddition of singlet oxygen to the Bz-His imidazole ring to give an unstable endoperoxide. This then underwent changes followed by nucleophilic addition and the elimination of one molecule of water to give 1.

The role of His 143 for the pH-dependent stability of plasminogen activator inhibitor-1

Using site-directed mutagenesis, His 143 on the α-helix F of PAI-1 was substituted by Lys, Asp, Phe and Thr, respectively. The generated single-site changed plasminogen activator inhibitor-1 (PAI-1) mutants were expressed in Escherichia coli and purified by heparin–Sepharose and anhydrotrypsin agarose chromatographies. When compared with wild-type (wtPAI-1), the PAI-1 mutants His143Asp and His143Phe had shorter half-lives at pH 7.5 (1.1 and 1.4 h, respectively, vs. 2 h for wtPAI-1). They also exhibited less pH dependency of their stability, with half-lives at pH 5.5 of 2.5 and 2.2 h, respectively, vs. 18 h for wtPAI-1. However, the PAI-1 mutants His143Lys and His143Thr had similar properties as wtPAI-1 in this respect. In conclusion, our results suggest that His 143 in one way or another might be involved in the pH-dependent stability of PAI-1. However, it seems that the protonation of this particular residue is of less importance. The PAI-1 mutants His143Asp and His143Phe only displayed about 20% of the specific activity obtained for wtPAI-1, because they, to a large extent, act as substrates for tissue-type plasminogen activator.

Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase/oxygenase.

Active-site His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3-hydroxyl of bound substrate or reaction-intermediate analogue (CABP), water molecules, and ligands for the activator metal-ion (Andersson I, 1996, J Mol Biol 259:160-174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432-444). To test structure-based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild-type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (approximately 10(3)-fold and > 10(5)-fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10-fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward-processing of carboxylated intermediate.

The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop.

A histidine residue with a pKa of 7 has been inferred to act as a general acid-base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochondrial isoenzyme Mib-CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small-angle X-ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild-type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters K(m) or Vmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.

Ribonuclease A mutant His 119Asn: the role of histidine in catalysis

Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His 12, His 119, and Lys 41) are known to be involved in catalysis. Mutation of His 119 to asparagine was generated to study the role of His 119 in RNase A catalysis. The mutant enzyme has been isolated and characterized. The mutation significantly decreases the rate of the transesterification reaction and has no effect on substrate affinity of the enzyme. An analysis of the enzymatic properties of H119N RNase A suggests that the imidazole ring of His 119 of the wild-type enzyme must be protonated in an enzyme-substrate productive complex. Thus our results indicate that a contribution of protonated His 119 into the catalysis is not restricted to protonation of oxygen atom of the substrate leaving group and that His 119 participates directly in a transition state stabilization via hydrogen bonding.