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A T Smith

Publications and source records attributed to A T Smith.

At least 55 records · Page 3Linked to original sources
PubMed1997

Identification of a critical phenylalanine residue in horseradish peroxidase, Phe179, by site-directed mutagenesis and 1H-NMR: implications for complex formation with aromatic donor molecules.

The functional and structural significance of Phe179 of horseradish peroxidase isoenzyme C (HRP C) has been investigated by site-directed mutagenesis. This residue is located in a structurally variable insertion between helices F and G, a motif unique to peroxidases of higher plants. Results obtained for three recombinant enzymes, with Phe179 substituted by Ala, His, or Ser, provide the first demonstration of the importance of this side chain for the binding of aromatic donor molecules. Experimental parameters for direct comparison with the wild-type enzyme were obtained by extensive solution state characterization using both optical and 1H-NMR spectroscopy. Significant chemical shift variations for resonances associated with the exposed heme edge, notably heme methyl C18H3 and heme propionate C17(1)H2, were recorded in NMR spectra of both the resting and cyanide-ligated states of the three Phe179 mutants. Furthermore, comparison of NOE connectivities in NOESY spectra of cyanide-ligated wild-type and mutant enzymes enabled the elusive assignment of the aromatic side chain in close proximity to heme methyl C18H3 to be made to Phe179. Replacement of Phe179 by Ala resulted in an 80-fold decrease in the binding affinity of the cyanide-ligated enzyme for benzhydroxamic acid, with a Kd value similar to that determined for cyanide-ligated HRP A2 (an acidic isoenzyme with valine at position 179). The binding affinity of Phe179-->Ser was similarly decreased, while that of Phe179-->His was partially restored relative to wild-type HRP C. Cyanide-ligated Phe179-->His HRP C exhibited a unique pH-dependent spectral transition associated with a pKa value of 6.5 +/- 0.2, assigned to the His179 side chain. Two closely related enzyme forms exhibiting different affinities for benzhydroxamic acid were observed at neutral pH and above, indicating that the protonation state of His179 gave rise to microheterogeneity in the aromatic donor molecule binding site.

Benzoates↗
PubMed1997

Chemical, spectroscopic and structural investigation of the substrate-binding site in ascorbate peroxidase.

The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant. Kd, of 11.6 +/- 0.4 microM (pH 7.002, mu = 0.10 M, 25.0 degrees C). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular-modelling studies, using the NNM-derived distance restraints in conjunction with the crystal structure of the enzyme [Patterson, W. R. & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated.

Ascorbate Peroxidases↗
PubMed1997

pH dependence and structural interpretation of the reactions of Coprinus cinereus peroxidase with hydrogen peroxide, ferulic acid, and 2,2'-azinobis.

Steady-state and transient-state analysis of Coprinus cinereus peroxidase, CIP (identical to Arthromyces ramosus peroxidase), was used to characterize the kinetics of the three fundamental steps in heme peroxidase catalysis: compound I (cpd I) formation, cpd I reduction, and compound II (cpd II) reduction. The rate constant k1 for cpd I formation determined by transient-state analysis is (9.9 +/- 0.6) x 10(6) M-1 s-1. The k1 determined by steady-state analysis is (8.8 +/- 0.6) x 10(6) M-1 s-1 in the presence of ferulic acid and (6.7 +/- 0.2) x 10(6) M-1 s-1 in the presence of ABTS. The value of k1 is constant from pH 6 to 11. However, at low pH the value of k1 decreases, corresponding to titration of an enzyme group with a pKa of 5.0. Titration of this group is also detected from cyanide-binding kinetics. Furthermore, titration of this group is linked with marked spectroscopic changes unique to CIP. We ascribe these changes to protonation of proximal His183. A very low pKa is proposed for distal His55 in the resting state of CIP. The rate constants, k2 for cpd I and k3 for cpd II reduction, are very large for both ferulic acid and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). For ferulic acid, transient-state kinetic analysis shows that the values of k2 and k3 are identical at pH 5-6, and the ratio k2/k3 increases to 10 at pH 10. The similar magnitude of k2 and k3 is unusual for a peroxidase. Both k2 and k3 decrease with increasing pH, and both are influenced by two ionizations: one with a pKa value near 7, assumed to reflect the protonation of His55; and the other with pKa of 9.0 +/- 0.7 for k2 and 8.8 +/- 0.4 for k3, perhaps reflecting the phenol-linked deprotonation of ferulic acid. Steady-state analysis at pH 7.0 gave k2k3/(k2 + k3) = (2.2 +/- 0.1) x 10(7) M-1 s-1 for ferulic acid, and (2.0 +/- 0.7) x 10(7) M-1 s-1 for ABTS and revealed a unimolecular step with ku = 1500 s-1, ascribed to slow ABTS radical product release. From transient-state results at pH 7, the values of k2 and k3 were found to be identical also for ABTS. A mechanism for cpd I and II reduction involving distal histidine and arginine is proposed.

Benzothiazoles↗
PubMed1997

Mutation of distal residues of horseradish peroxidase: influence on substrate binding and cavity properties.

The manner in which the distal heme pocket residues of peroxidases control the reaction mechanism and ligand binding has been investigated further by analysis of the electronic absorption and resonance Raman (RR) spectra of distal site mutants of recombinant horseradish peroxidase (HRP-C*). The roles of the conserved distal histidine and arginine residues, particularly in the context of the catalytic mechanism originally proposed for cytochrome c peroxidase (CCP), have been evaluated by studying the His42 --> Leu, His42 --> Arg, Arg38 --> Gly, and Arg38 --> Leu variants of HRP-C*. Spectra of the ferric forms, their complexes with benzohydroxamic acid (BHA), and the ferrous forms have been recorded at neutral pH. In addition, the ferric forms have been studied at alkaline pH. The relative populations of the three heme spin states characteristic of HRP-C* and its mutants were found to vary markedly from mutant to mutant. This diversity of heme spin state populations among the various mutants has allowed a well-defined set of RR frequencies to be compiled for the three heme spin states. These frequencies support the analysis of wild-type HRP-C* in terms of two heme states, five- (5cHS#) and six-coordinate high-spin (6cHS#), which exhibit anomalous RR frequencies compared to those of model heme systems. The third heme spin state is identified as being six-coordinate high-spin, displaying typical RR frequencies (6cHS). The 6cHS# and the 6cHS heme states are characterized by H bonding between the iron-bound water molecule and the Arg38 residue or the His42 residue, respectively. The proportion of six-coordinate high-spin heme states is at a minimum in the Arg38Leu mutant, indicating that the occupancy of the distal water molecule site is reduced in this mutant. The His42Arg mutant is distinguished from the other mutants by the unexpected presence of an iron-bound hydroxyl group at neutral pH. The spectral changes induced upon complexation with BHA indicate that both the distal histidine and arginine are involved in BHA binding; however, the arginine residue appears to play a more critical role. Measurements at pH 12 suggest there is a concerted involvement of both distal residues in mediating the alkaline transition of HRP-C*. Arg38 appears to be essential for stabilization of the OH- ligand, while His42 acts as a H bond acceptor. A striking similarity between the roles of these residues in the reaction of H2O2 with the enzyme and the alkaline transition is noted. By comparison with the results from corresponding mutants of CCP, it appears that although the hydrogen-bonding network linking the distal and proximal sides of the heme is conserved the distal cavity in HRP-C differs significantly from that of CCP. However, some similarities in the local environment of the distal arginine are suggested.

Crystallography, X-Ray↗
PubMed1997

Detection and discrimination of first- and second-order motion in patients with unilateral brain damage.

The present investigation explored the extent to which extrastriate cortex is necessary for various aspects of motion processing and whether the processing of first-order (Fourier) and second-order (non-Fourier) motion involves the same extrastriate cortical regions. Orientation, direction, and speed discrimination thresholds were measured in 21 patients with unilateral damage to the lateral occipital, temporal, or posterior parietal cortex. Their results were compared with those of 14 age-matched control subjects. The stimuli were static random-dot noise patterns, the luminance of which (first-order) or contrast (second-order) was modulated by a drifting sinusoid. Each image was presented at an eccentricity of 5.6 deg in one of the four visual quadrants. The contrasts required to identify orientation and direction were measured in a forced-choice paradigm for three speeds (1.5, 3, and 6 deg/sec). Speed discrimination performance was measured for stimuli presented simultaneously in two of the four quadrants. The results indicate the following: (1) orientation thresholds were increased only slightly in the patients; (2) direction thresholds were modestly elevated, and this effect was more pronounced for second-order stimuli than for first-order stimuli; (3) speed discrimination thresholds were elevated significantly in the patients with lesions in the region bordering superior-temporal and lateral-occipital cortex; and (4) speed discrimination thresholds for first-order stimuli were more elevated than those for second-order stimuli. The results suggest that there is substantial overlap in the cortical areas involved in first- and second-order speed discrimination.

Adult↗
PubMed1997

Effect of distal cavity mutations on the binding and activation of oxygen by ferrous horseradish peroxidase.

Mutations have been introduced at residues Arg-38 or His-42 in horseradish peroxidase isoenzyme C (HRPC) in order to probe the role of these key distal residues in the reaction of ferrous HRPC with dioxygen. The association and dissociation rate constants for dioxygen binding to His-42 --> Leu, His-42 --> Arg, Arg-38 --> Leu, Arg-38 --> Lys, Arg-38 --> Ser, and Arg-38 --> Gly variants have been measured using stopped-flow spectrophotometry. Replacement of His-42 by Leu or Arg increases the oxygen binding rate constant by less than an order of magnitude, whereas changing the polar distal Arg-38 causes increases of more than 2 orders. These results demonstrate that His-42 and Arg-38 impede the binding of dioxygen to ferrous HRPC, presumably by steric and/or electrostatic interactions in the distal heme cavity. Recombinant HRPC oxyperoxidase reverted slowly to the ferric state with no spectrophotometrically detectable intermediates and with an apparent first-order rate constant of 9.0 x 10(-3) s(-1), which is essentially the same as that for the native, glycosylated enzyme. This reaction was accelerated when His-42 was replaced by Leu or Arg (k(decay) = 0.10 and 0.07 s(-1), respectively) presumably due to the loss of the hydrogen bond between the His-42 imidazole and the bound dioxygen. Substitution of Arg-38 by Leu, Lys, or Gly also produced a less stable oxyperoxidase (k(decay) = 0.22, 0.20, and 0.58 s(-1), respectively). However, with the Arg-38 --> Ser variant, a transient intermediate, proposed to be a ferric-superoxide complex, was detected by rapid-scan stopped-flow spectrophotometry during the conversion of oxyperoxidase to the ferric state. This variant also exhibits an unusually high affinity for dioxygen. It is proposed that Arg-38 interacts with the bound dioxygen to promote superoxide character, thereby stabilizing the oxyperoxidase state and making the binding of dioxygen to ferrous HRPC essentially irreversible. We conclude that Arg-38 and His-42 not only promote the heterolytic cleavage of bound hydrogen peroxide to form compound I but also decrease the lability of the ferrous enzyme-dioxygen complex in order to suppress the formation of the inactive ferrous state.

Arginine↗
PubMed1997

Changes in perceived speed following adaptation to first-order and second-order motion.

To investigate whether or not adaptation to second-order motion can cause changes in perceived speed, measurements of perceived speed were obtained for two varieties of motion: (i) contrast-modulated two-dimensional static noise (second-order motion); and (ii) luminance-modulated noise (first-order motion). The test stimulus (either first-order or second-order) was presented to one side of a central fixation spot and a comparison stimulus (always first-order) was simultaneously presented on the opposite side. The observer's task was to indicate which of the two motion stimuli appeared to drift faster. The perceived speed of the test stimulus was measured with and without prior adaptation to motion on one side of the fixation spot only (that of the test stimulus). The modulation depth of the adaptation stimulus was always half that of the test stimulus and all test patterns were equated for visibility. The pattern of results for second-order motion was similar to that for first-order motion. Typically, adaptation reduced perceived speed, particularly when the adaptation speed was faster than the test speed. However, when the adaptation speed was low relative to the test speed, increases in perceived speed were found. Cross-over adaptation effects between first-order and second-order motion were also observed. Robust velocity aftereffects were found for second-order motion when the noise was dynamic or was high-pass filtered, suggesting that first-order (luminance) artifacts were not responsible for the velocity aftereffects observed. We conclude that the perceived speeds of first-order and second-order motion appear to be encoded in human vision using similar computational principles (but not necessarily utilizing the same mechanism), since the same pattern of results was found for the two varieties of motion.

Adaptation, Physiological↗
PubMed1997

Separate detection of moving luminance and contrast modulations: fact or artifact?

We have investigated first-order artifacts in second-order motion perception. Subjects were required to identify the orientation and direction of a drifting sinusoidal contrast modulation. When the carrier consisted of static two-dimensional noise, performance often reflected the use of first-order artifacts that arise from stochastic local biases in the noise, rather than the detection of the contrast modulation per se. This stimulus, which has been used widely for studying second-order motion, therefore appears to be inappropriate for that purpose. In contrast, global distortion products arising from luminance non-linearities do not appear to provide usable artifacts. Two manipulations were employed to eliminate local first-order artifacts: the use of dynamic noise and the use of high-pass filtered static noise. These two manipulations gave similar results, which were quite different from those obtained with broadband static noise. We argue that performance with both of these image types reflects the activity of a true second-order motion mechanism. A characteristic property of this mechanism is that it cannot specify direction at the threshold for detecting orientation. Direction thresholds are around 50% higher than orientation thresholds when first-order artifacts are eliminated.

Contrast Sensitivity↗
PubMed1997

Crystal structure of horseradish peroxidase C at 2.15 A resolution.

The crystal structure of horseradish peroxidase isozyme C (HRPC) has been solved to 2.15 A resolution. An important feature unique to the class III peroxidases is a long insertion, 34 residues in HRPC, between helices F and G. This region, which defines part of the substrate access channel, is not present in the core conserved fold typical of peroxidases from classes I and II. Comparison of HRPC and peanut peroxidase (PNP), the only other class III (higher plant) peroxidase for which an X-ray structure has been completed, reveals that the structure in this region is highly variable even within class III. For peroxidases of the HRPC type, characterized by a larger FG insertion (seven residues relative to PNP) and a shorter F' helix, we have identified the key residue involved in direct interactions with aromatic donor molecules. HRPC is unique in having a ring of three peripheral Phe residues, 142, 68 and 179. These guard the entrance to the exposed haem edge. We predict that this aromatic region is important for the ability of HRPC to bind aromatic substrates.

Amino Acid Sequence↗
PubMed1997

Nonrandom mating in pikas Ochotona princeps: evidence for inbreeding between individuals of intermediate relatedness.

The extent and effect of inbreeding in natural populations remain largely undetermined. Pikas Ochotona princeps have been considered a likely candidate for close inbreeding in natural populations due to observations of frequent juvenile philopatry (colonization of natal home range or neighbouring home range) and high levels of spatial overlap and social tolerance between neighbouring individuals of the opposite sex. A 4-year investigation of inbreeding in pikas, however, has revealed that dispersal and mating patterns are uncoupled in this species, i.e. explained by different hypotheses. DNA fingerprinting analysis revealed that band-sharing scores between mated pairs, identified via parentage analysis, were not commensurate with band-sharing among known first-order relatives, but were similar to scores for a small sample of known second-order relatives (i.e. half-siblings grandparent-grandchild pairs). Band-sharing scores between mated pairs were then compared with those between potential mated pairs within the population to assess whether mating was random or nonrandom with respect to genetic similarity. The results of Monte Carlo randomization tests show that pikas mated with individuals with intermediate genetic similarity in greater proportion than would be expected by chance. These data suggest mate choice in pikas may be based upon intermediate levels of relatedness.

Animals↗
PubMed1996

Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and haem.

An engineered cDNA from Phanerochaete chrysosporium encoding both the mature and pro-sequence regions of Lip isoenzyme H8 (Lip) has been successfully overexpressed in Escherichia coli. The recombinant protein (LipP*) was sequestered in inclusion bodies. The reduced-denatured polypeptide has been purified by differential solubilization, and the active enzyme recovered after controlled in vitro refolding (albeit in low yield), by glutathione-mediated oxidation of disulphides, in a folding medium containing an intermediate concentration of urea, Ca2+, and haem. The procedure is analogous to that previously described for the production of active recombinant horseradish peroxidase (HRP-C*) from inclusion-body material. It is quite possible, therefore, that this type of procedure may be suitable for the recovery of most, if not all, active recombinant peroxidases. The resultant LipP* has spectral characteristics identical with that of the native enzyme as isolated from Phanerochaete chrysosporium. Its specific activity measured in the standard veratryl alcohol (VA) assay was 39 micromol of VA oxidized/min per mg of protein, a value which compares extremely favourably with that of the native enzyme (36 micromol of VA/min per mg). Although levels of active enzyme obtained are not yet as high as in the case of HRP-C* (1% conversion of crude inactive LipP* polypeptide into pure fully active Lip), it is envisaged that further refinement of the expression/folding/activation procedures will provide sufficient protein for biophysical characterization of both the wild-type and site-directed mutants.

Base Sequence↗
PubMed1996

Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine.

The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus peroxidase was more resistant to haem modification and phenyl-radical-based inactivation than HRP-C.

Binding Sites↗
PubMed1996

Role of arginine 38 in horseradish peroxidase. A critical residue for substrate binding and catalysis.

The observed pseudo-first order rate constant for the reaction between a horseradish peroxidase (HRP) variant (R38L)HRPC* and hydrogen peroxide saturates at high peroxide concentrations (Km = 11. 8 mm). The data are consistent with a two-step mechanism involving the formation of an HRP-H2O2 intermediate (k = 1.1 x 10(4) m-1 s-1) whose conversion to compound I is rate-limiting (k = 142 s-1) suggesting that Arg-38 is not only involved in the cleavage of the O-O bond of peroxide but also has an important role in facilitating the rapid binding of H2O2 to HRP. Rapid-scan spectrophotometry revealed the presence of a transient intermediate with a spectrum consistent with a ferric-hydroperoxy complex. At high peroxide concentrations (>500 microM), compound I is converted to compound III without the accumulation of compound II. Spectrophotometric titrations show that arginine 38 is also involved in modulating the apparent affinity of HRPC for reducing substrates such as guaiacol and p-cresol. The spectrum of the complex formed when these substrates bind to the ferric form of the mutant enzyme differs from that observed when they bind to the wild-type ferric enzyme. At neutral and alkaline pH compound I of (R38L)HRPC* was stable and reduced to ferric enzyme without apparent formation of compound II upon titration with p-cresol or ascorbic acid, suggesting a change in the rate-limiting step in the peroxidase cycle. Steady-state kinetic analyses carried out at pH 7.0 showed significant increases in the apparent Km for guaiacol, p-cresol, and 2, 2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS). The high stability of the oxyferryl form of (R38L)HRPC* and its low catalytic constant for reducing substrates also shows that arginine 38 modulates the reactivity of HRP compound I.

Amino Acid Sequence↗
PubMed1996

Refinement of 3D models of horseradish peroxidase isoenzyme C: predictions of 2D NMR assignments and substrate binding sites.

In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)-differing mainly in the structure of a long untemplated insertion-were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca2+ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures.

Amino Acid Sequence↗
PubMed1995

A comparative study of the inactivation of wild-type, recombinant and two mutant horseradish peroxidase isoenzymes C by hydrogen peroxide and m-chloroperoxybenzoic acid.

The mechanism-based inactivation of four horseradish peroxidase (HRP-C) enzyme variants has been studied kinetically with either hydrogen peroxide or the xenobiotic m-chloroperoxybenzoic acid (mClO2-BzOH) as sole substrate. The concentration and time dependence of inactivation was investigated for the wild-type plant enzyme (HRP-C), the unglycosylated recombinant enzyme (HRP-C*), and two site-directed mutants with Phe143 replaced by Ala ([F143A]HRP-C*) or Arg38 replaced by Lys ([R38K]HRP-C*). The number of turnovers (r) of H2O2 required to completely inactivate the enzymes was found to vary between the different enzymes with HRP-C being most resistant to inactivation (r = 625), HRP-C* and [F143A]HRP-C* being approximately twice as sensitive (r = 335 and 385, respectively) in comparison, and [R38K]HRP-C* being inactivated much more easily (r = 20). In the cases of HRP-C* and [F143A]HRP-C*, compared to HRP-C the differences were due to the absence of glycosylation on the exterior of the proteins, whilst the [R38K]HRP-C* variant exhibited a distinct mechanistic difference. When mClO2BzOH was used as the substrate the differences in sensitivity to inactivation disappeared. The values of r were all around 3 reflecting the strong affinity of mClO2BzOH for the active site. The apparent rate constant for inactivation by H2O2 was found to be about twofold higher in [R38K]HRP-C* than the other enzymes and the catalytic constant for turnover of H2O2 was approximately ten times lower. The affinity of compound I for H2O2 leading to the formation of a transitory intermediate implicated in the inactivation of peroxidase decreased in the order HRP-C, HRP-C*, [F143A]HRP-C*, [R38K]HRP-C*.

Binding Sites↗
PubMed1995

Laser photolysis behavior of ferrous horseradish peroxidase with carbon monoxide and cyanide: effects of mutations in the distal heme pocket.

Native horseradish peroxidase and several forms with mutations in the distal heme pocket (His42Leu, His42Arg, and Arg38Leu) have been expressed in Escherichia coli. These enzymes have been purified and analyzed in terms of the room temperature recombination rate of carbon monoxide and cyanide after photolysis of the reduced forms. The recombinant wild-type ferrous form exhibited monophasic recombination of carbon monoxide with an observed bimolecular rate constant at pH 8.5 of 4.4 x 10(3) M-1 s-1 which is essentially the same as the natural glycosylated form. This recombination rate constant increases in the mutants in the order WT < H42R < H42L << R38L. The value for R38L (5 x 10(6) M-1 s-1) is increased by 3 orders of magnitude relative to the wild-type and is similar to that for human hemoglobin [Mims et al. (1983) J. Biol. Chem. 258, 14219-14232]. Cyanide recombination with the wild-type ferrous form at room temperature is biphasic at pH 6.5 but becomes more monophasic at pH 8.5, again similar to the behavior of the natural glycosylated form, although the Fe(2+)-cyano form of the recombinant enzyme appears to be more unstable at high pH. None of the mutant forms were able to bind cyanide in the ferrous state to any significant extent (Kdiss > 250 mM) when cyanide was added at a concentration (10-20 mM) sufficient to almost saturate the wild-type form (Kdiss approximately equal to 1 mM at pH 7). This behavior contrasts with that of the oxidized forms of the mutants where increases in cyanide dissociation constants are smaller ( < 25 times).(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1995

Solution characterisation by NMR spectroscopy of two horseradish peroxidase isoenzyme C mutants with alanine replacing either Phe142 or Phe143.

Site-directed mutagenesis of the horseradish peroxidase isoenzyme C (HRP C) gene has been undertaken in order to provide two recombinant enzymes where alanine replaces either Phe142 or Phe143 ([F142A]HRP C and [F143A]HRP C, respectively). These heme enzymes have been characterised in solution using proton NMR spectroscopy for both the high-spin resting and low-spin cyanide-ligated states. Comparison of their NMR spectra with those recorded for wild-type plant HRP C indicates that both the protein fold and the structure of the heme pocket are maintained. The structural integrity of the aromatic donor molecule binding site is altered as a result of the substitution of Phe142 by Ala, but not by the corresponding substitution at Phe143. This is evident from analysis of perturbations to the chemical shift and linewidth parameters of the proton resonances of two Phe side chains, Phe A and Phe B, that participate in this site. The resting and cyanide-ligated states of [F142A]HRP C bind the aromatic donor molecule, benzhydroxamic acid, three to four times more weakly than the analogous states of wild-type plant HRP C. A titration of cyanide-ligated [F142A]HRP C with benzhydroxamic acid, monitored by NMR spectroscopy, further reveals that the dynamics of complex formation are considerably altered, in that only one of the two possible benzhydroxamic acid binding modes established for the cyanide-ligated wild-type enzyme is significantly populated. Although the assignment of Phe A and Phe B cannot be made to either Phe142 or Phe143, the results confirm that Phe142 is an important, although indirect, determinant of aromatic donor molecular binding and dynamics. The role of phenylalanine side chains in the binding of aromatic donor molecules by heme peroxidases is discussed in the light of these observations and a recent structural model for HRP C.

Alanine↗