Biomedical subjects

J Kraut

Publications and source records attributed to J Kraut.

At least 55 records · Page 3Linked to original sources

PubMed1991

Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding.

The crystal structure of unliganded dihydrofolate reductase (DHFR) from Escherichia coli has been solved and refined to an R factor of 19% at 2.3-A resolution in a crystal form that is nonisomorphous with each of the previously reported E. coli DHFR crystal structures [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, B. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662; Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277]. Significant conformational changes occur between the apoenzyme and each of the complexes: the NADP+ holoenzyme, the folate-NADP+ ternary complex, and the methotrexate (MTX) binary complex. The changes are small, with the largest about 3 A and most of them less than 1 A. For simplicity a two-domain description is adopted in which one domain contains the NADP+ 2'-phosphate binding site and the binding sites for the rest of the coenzyme and for the substrate lie between the two domains. Binding of either NADP+ or MTX induces a closing of the PABG-binding cleft and realignment of alpha-helices C and F which bind the pyrophosphate of the coenzyme. Formation of the ternary complex from the holoenzyme does not involve further relative domain shifts but does involve a shift of alpha-helix B and a floppy loop (the Met-20 loop) that precedes alpha B. These observations suggest a mechanism for cooperativity in binding between substrate and coenzyme wherein the greatest degree of cooperativity is expressed in the transition-state complex. We explore the idea that the MTX binary complex in some ways resembles the transition-state complex.

Amino Acid Sequence↗

PubMed1991

Compound I radical in site-directed mutants of cytochrome c peroxidase as probed by electron paramagnetic resonance and electron-nuclear double resonance.

The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Electron redistribution on binding of a substrate to an enzyme: folate and dihydrofolate reductase.

The migration of electron density of a substrate (folate) on binding to an enzyme (dihydrofolate reductase) is studied by a quantum-mechanical method originally developed in solid state physics. A significant polarization of the substrate is induced by the enzyme, toward the transition state of the enzymatic reaction, at the same time giving rise to "electronic strain energy" in the substrate and enhanced protein-ligand interactions. The spatial arrangement of protein charges that induces the polarization is identified and found to be structurally conserved for bacterial and vertebrate dihydrofolate reductases.

Binding Sites↗

PubMed1991

The electrostatic potential of Escherichia coli dihydrofolate reductase.

Escherichia coli dihydrofolate reductase (DHFR) carries a net charge of -10 electrons yet it binds ligands with net charges of -4 (NADPH) and -2 (folate or dihydrofolate). Evaluation and analysis of the electrostatic potential of the enzyme give insight as to how this is accomplished. The results show that the enzyme is covered by an overall negative potential (as expected) except for the ligand binding sites, which are located inside "pockets" of positive potential that enable the enzyme to bind the negatively charged ligands. The electrostatic potential can be related to the asymmetric distribution of charged residues in the enzyme. The asymmetric charge distribution, along with the dielectric boundary that occurs at the solvent-protein interface, is analogous to the situation occurring in superoxide dismutase. Thus DHFR is another case where the shape of the active site focuses electric fields out into solution. The positive electrostatic potential at the entrance of the ligand binding site in E. coli DHFR is shown to be a direct consequence of the presence of three positively charged residues at positions 32, 52, and 57--residues which have also been shown recently to contribute significantly to electronic polarization of the ligand folate. The latter has been postulated to be involved in the catalytic process. A similar structural motif of three positively charged amino acids that gives rise to a positive potential at the entrance to the active site is also found in DHFR from chicken liver, and is suggested to be a common feature in DHFRs from many species. It is noted that, although the net charges of DHFRs from different species vary from +3 to -10, the enzymes are able to bind the same negatively charged ligands, and perform the same catalytic function.

Amino Acids↗

PubMed1991

Changes in the electron density of the cofactor NADPH on binding to E. coli dihydrofolate reductase.

Quantum-mechanical electron density calculations reveal that a significant polarization is induced in the cofactor NADPH (reduced nicotinamide adenine dinucleotide phosphate) on binding to the enzyme dihydrofolate reductase. The calculations indicate that electron density corresponding to approximately 0.7 electron charges is shifted within the molecule, extending over more than 20 A. Further calculations on proposed enzyme mutants show that the polarization of NADPH on binding to DHFR is, in large part, induced by a motif of three positively charged residues. This motif was also identified to be directly responsible for the positive electrostatic potential surrounding the cofactor binding site in the enzyme. The possibility of this long-range polarization of NADPH was originally proposed based on a previous study of ligand binding to DHFR where a conserved structural motif of three positively charged residues was found to play a major role in polarizing the substrate folate over its entire length of 18 A.

Arginine↗

PubMed1991

Temafloxacin pharmacokinetics in subjects with normal and impaired renal function.

The pharmacokinetics of temafloxacin were investigated following oral administration of single 400-mg doses to 6 normal subjects and 18 subjects with various degrees of impaired renal function. Renal impairment did not significantly affect the peak concentration, time to peak concentration, or the nonrenal clearance of temafloxacin. Both renal clearance (CLR) and total apparent clearance (CLT/F, where F represents the fraction of dose absorbed) of temafloxacin were highly correlated with creatinine clearance (CLCR). The regression equations were as follows: CLR = 0.85.CLCR, with R2 = 0.907, and CLT/F = 56.0 + 0.92.CLCR, with R2 = 0.656. The half-life (mean +/- standard deviation) increased from 10.6 +/- 2.4 h in the normal volunteers to 24.6 +/- 7.3 h in the subjects with a CLCR of less than 10 ml/min; the respective CLT/F decreased from 169 +/- 58 to 70 +/- 27 ml/min. Compared with the CLT/F in the subjects with normal renal function, CLT/F was reduced 60% in subjects with a CLCR of less than 40 ml/min, indicating that the dosage should be reduced by at least one-half for patients with comparable impairment. For the subjects on chronic hemodialysis, most of the variability in the nonrenal clearance and the terminal-phase rate constant of temafloxacin was associated with the quantity of calcium carbonate and related medication taken for the treatment of hyperphosphatemia. Supplemental dosage is not required for patients undergoing hemodialysis, since the distribution of temafloxacin in tissue is extensive and the recoveries from 4-h dialysis sessions accounted for less than 10% of the drug present at the start of the dialysis.

Adult↗

PubMed1991

Siderophore presence in sputa of cystic fibrosis patients.

Sputum samples from the lungs of cystic fibrosis patients harboring Pseudomonas aeruginosa infections were collected and examined for the presence of the siderophore pyoverdine. Fluorescence quenching, due to the addition of ferric ion, as well as column and thin-layer chromatography results indicated that all samples contained the siderophore. Six samples furnished sufficient material after purification to allow us to obtain visible absorbance spectra. These spectra were characteristic of the ferrated analog of the P. aeruginosa pyoverdine, that is, ferripyoverdine, and in all cases they indicated a degree of ferration in excess of 50%. P. aeruginosa in the cystic fibrosis lung is thus iron stressed and responds by synthesizing pyoverdine, which subsequently binds ferric ion.

Adolescent↗

PubMed1991

Laboratory evaluations on thermal debonding of ceramic brackets.

The purpose of this laboratory study was to define the working parameters and physiological safety and efficacy of the Dentaurum Ceramic Debonding Unit. Extracted mandibular incisors were utilized because of their low thermal mass and low heat sensitivity. The teeth were embedded in plastic and placed on a turning force measuring apparatus. An electrothermal element was placed in the pulp chamber (filled with a conducting paste). The thermoelement temperature was registered on y-t recorder as was the turning momentum required to remove the ceramic brackets with the Dentaurum Ceramic Debonding Unit. Ceramic brackets from GAC (Allure III), Unitek (Transcend) and Dentaurum (Fascination) using one and two component adhesives (Monolok, Concise), were tested. Scanning electron microscopic views taken after debonding showed predictable (and favorable) adhesive failure at the bracket base/resin interface. No enamel damage was demonstrated. All brackets were removable under three seconds with a clinically reproducible turning force of 85-100 Nmm allowing for intrapulpal temperature increases under the 5 degrees C biocompatible threshold. The Dentaurum Ceramic Debonding Unit provided a safe, reliable, efficient modality of removing ceramic brackets while maintaining a physiologically acceptable rise in pulpal temperature without damage to tooth enamel or pulpal tissue.

Body Temperature↗

PubMed1991

Clinical evaluations on thermal versus mechanical debonding of ceramic brackets.

Removal of ceramic orthodontic brackets, utilizing orthodontic pliers, has resulted in significant patient discomfort, enamel trauma and bracket shattering. The purpose of this clinical study was to compare the safety and efficacy of standard orthodontic mechanical debonding with a new Dentaurum thermal debonding device. Fifteen healthy patients with maxillary or mandibular premolars scheduled for extraction completed this study. One week after bracket placement, removal was accomplished by mechanical pliers or a thermal debonding device. Both procedures were performed on each patient in random order. Efficacy was monitored using scanning electron microscopy (SEM) on impressions made before and at selected times after debonding. Extracted teeth were subjected to histological evaluation to check potential injury to the pulp. Comfort levels were determined by questionnaires. Data was analyzed by Chi square. No irritation or overt changes were reported or observed. Significant differences (p less than 0.05) favoring the thermal devices were determined from the questionnaire on comfort during the removal process. Evaluation of SEM surfaces found significantly less (p less than 0.05) changes produced with the thermal debonding device, compared to mechanical removal. No histological evidence of pulpal injury related to product treatments were determined. The Dentaurum thermal debonding device was judged less traumatic by patients and produced minimal enamel surface changes compared to a mechanical debonding plier.

Adolescent↗

PubMed1990

CO dissociation in cytochrome c peroxidase: site-directed mutagenesis shows that distal Arg 48 influences CO dissociation rates.

To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast [CCP(MI)] and in several mutants in the heme binding pocket prepared by site-directed mutagenesis. The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly. Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form. Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here. In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI). Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly. In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms. Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗

PubMed1990

Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate.

The 2.3-A crystal structure of recombinant human dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a binary complex with folate (a poor substrate at neutral pH) and also as a binary complex with an inhibitor, 5-deazafolate. The inhibitor appears to be protonated at N8 on binding, whereas folate is not. Rotation of the peptide plane joining I7 and V8 from its position in the folate complex permits hydrogen bonding of 5-deazafolate's protonated N8 to the backbone carbonyl of I7, thus contributing to the enzyme's greater affinity for 5-deazafolate than for folate. In this respect it is likely that bound 5-deazafolate furnishes a model for 7,8-dihydrofolate binding and, in addition, resembles the transition state for folate reduction. A hypothetical transition-state model for folate reduction, generated by superposition of the DHFR binary complexes human.5-deazafolate and chicken liver.NADPH, reveals a 1-A overlap of the binding sites for folate's pteridine ring and the dihydronicotinamide ring of NADPH. It is proposed that this binding-site overlap accelerates the reduction of both folate and 7,8-dihydrofolate by simultaneously binding substrate and cofactor with a sub van der Waals separation that is optimal for hydride transfer.

Binding Sites↗

PubMed1990

A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase.

The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.

Bacterial Proteins↗

PubMed1990

Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235----Asn-235 mutant.

Proton NMR spectra of cytochrome c peroxidase (CcP) isolated from yeast (wild type) and two Escherichia coli expressed proteins, the parent expressed protein [CcP(MI)] and the site-directed mutant CcP(MI,D235N) (Asp-235----Asn-235), have been examined. At neutral pH and in the presence of only potassium phosphate buffer and potassium nitrate, wild-type Ccp and CcP(MI) demonstrate nearly identical spectra corresponding to normal (i.e., "unaged") high-spin ferric peroxidase. In contrast, the mutant protein displays a spectrum characteristic of a low-spin form, probably a result of hydroxide ligation. Asp-235 is hydrogen-bonded to the proximal heme ligand, His-175. Changing Asp-235 to Asn results in alteration of the pK for formation of the basic form of CcP. Thus, changes in proximal side structure mediate the chemistry of the distal ligand binding site. All three proteins bind F-, N3-, and CN- ions, although the affinity of the mutant protein (D235N) for fluoride ion appears to be much higher than that of the other two proteins. Analysis of proton NMR spectra of the cyanide ligated forms leads to the conclusion that the mutant protein (D235N) possesses a more neutral proximal histidine imidazole ring than does either wild-type CcP or CcP(MI). It confirms that an important feature of the cytochrome c peroxidase structure is at least partial, and probably full, imidazolate character for the proximal histidine (His-175).

Asparagine↗

PubMed1990

X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis.

The 2.2-A X-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli [Fishel, L.A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360], has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53----Ile substitution in CCP(MI). A Trp-51----Phe mutant remains pentacoordinated and exhibits only minor distal structural adjustments. The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron [Smulevich, G., Mauro, J.M., Fishel, L. A., English, A. M., Kraut, J., & Spiro, T. G. (1988) Biochemistry 27, 5477-5485]. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191----Phe mutant that has substantially diminished enzyme activity and altered magnetic properties [Mauro, J. M., Fishel, L. F., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256] accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large (ca. 1 A) local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structure rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. Changing proximal Asp-235 to Asn results in two significant localized structural changes. First, the heme iron moves toward the porphyrin plane, and distal water 595 now clearly resides in the iron coordination sphere at a distance of 2.0 A. The observation of hexacoordinated iron for the D235N mutant is in accord with previous resonance Raman results. Second, the indole side chain of Trp-191 has flipped over as a result of the mutation; the tryptophan N epsilon takes part in a new hydrogen bond with the backbone carbonyl oxygen of Leu-177.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1990

Single-crystal resonance Raman spectroscopy of site-directed mutants of cytochrome c peroxidase.

Resonance Raman spectra are reported for single crystals of cytochrome c peroxidase (CCP) mutants, taken by using a microscope equipped with a variable-temperature stage. The spectra are similar to those observed for the mutant proteins in solution, but there are detectable differences having to do with the coordination and spin state of the heme. The Asn-235 mutant contains a mixture of six-coordinate high- and low-spin states with a detectably higher fraction of the former than in solution. Upon cooling even to 223 K, the heme is converted mostly to the low-spin form. The Phe-191 mutant likewise shows a high/low-spin six-coordinate mixture, together with a preponderant population of five-coordinate heme. Upon cooling, the high-spin six-coordinate population converts immediately to the low-spin form, while the five-coordinate population does so more slowly. This behavior is intermediate between that of native CCP and the Asn-235 mutant, consistent with an ancillary role for the normal Trp-191-Asp-235 H-bond in the proximal anchoring of the heme Fe. The Phe-51 mutant shows a dominant high-spin five-coordinate heme population in the single crystal, whereas in solution the six-coordinate form is dominant. This difference is mimicked by adding 2-methyl-2,4-pentanediol (MPD) to the solution and is attributed to the dehydrating effect of MPD, which is present during crystallization. Upon lowering the temperature, the five-coordinate heme converts partially to a six-coordinate high-spin form.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome-c Peroxidase↗

PubMed1990

Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH.

The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states, Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion of conformers are small (t1/2 for Ew----Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27 is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew----Et (t1/2 = 221 s) and an increased rate for Et----Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).

Aspartic Acid↗

PubMed1990

Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate.NADP+ ternary complex. Substrate binding and a model for the transition state.

The crystal structure of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli has been solved as the binary complex with NADP+ (the holoenzyme) and as the ternary complex with NADP+ and folate. The Bragg law resolutions of the structures are 2.4 and 2.5 A, respectively. The new crystal forms are nonisomorphous with each other and with the methotrexate binary complex reported earlier [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. In general, NADP+ and folate binding conform to predictions, but the nicotinamide moiety of NADP+ is disordered in the holoenzyme and ordered in the ternary complex. A mobile loop (residues 16-20) involved in binding the nicotinamide is also disordered in the holoenzyme. We report a detailed analysis of the binding interactions for both ligands, paying special attention to several apparently strained interactions that may favor the transition state for hydride transfer. Hypothetical models are presented for the binding of 7,8-dihydrofolate in the Michaelis complex and for the transition-state complex.

Catalysis↗

PubMed1990

Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants.

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.

Cytochrome-c Peroxidase↗