Transient-state Kinetic Studies Research Articles

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Quantifying the impact of initial RNA primer length on nucleotide addition by RNA polymerase I

Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous. This observation led to the question, what drives the variability of rate constants governing RNA synthesis by Pol I? Are the kinetics of nucleotide addition dictated by the position of the nascent RNA within the polymerase or by the identity of the next encoded nucleotide? In this study, we examine the impact of nucleotide position (i.e. nascent RNA primer length) on the rate constants governing nine sequential nucleotide addition events catalyzed by Pol I. The results reveal a conserved trend in the observed rate constants at each position for all primer lengths used, and highlight that the 9-nucleotide, or 9-mer, RNA primer provides the fastest observed rate constants. These findings suggest that the observed heterogeneity of rate constants for RNA synthesis by Pol I in vitro is driven primarily by the template sequence.

Transient-state kinetic analysis of complex formation between photoprotein clytin and GFP from jellyfish Clytia gregaria.

Luminous organisms use different protein-mediated strategies to modulate light emission color. Here, we report the transient-state kinetic studies of the interaction between photoprotein clytin from Clytia gregaria and its antenna protein, cgreGFP. We propose that cgreGFP forms a transient complex with Ca(2+)-bound clytin before the excited singlet state of the coelenteramide product is formed. From the spectral distribution and donor-acceptor separation distance, we infer that clytin reaction intermediates may interact only with the middle side part of cgreGFP.

Mechanistic and Kinetic Analysis of the DcpS Scavenger Decapping Enzyme

Decapping is an important process in the control of eukaryotic mRNA degradation. The scavenger decapping enzyme DcpS functions to clear the cell of cap structure following decay of the RNA body by catalyzing the hydrolysis of m(7)GpppN to m(7)Gp and ppN. Structural analysis has revealed that DcpS is a dimeric protein with a domain-swapped amino terminus. The protein dimer contains two cap binding/hydrolysis sites and displays a symmetric structure with both binding sites in the open conformation in the ligand-free state and an asymmetric conformation with one site open and one site closed in the ligand-bound state. The structural data are suggestive of a dynamic decapping mechanism where each monomer could alternate between an open and closed state. Using transient state kinetic studies, we show that both the rate-limiting step and rate of decapping are regulated by cap substrate. A regulatory mechanism is established by the intrinsic domain-swapped structure of the DcpS dimer such that the decapping reaction is very efficient at low cap substrate concentrations yet regulated with excess cap substrate. These data provide biochemical evidence to verify experimentally a dynamic and mutually exclusive cap hydrolysis activity of the two cap binding sites of DcpS and provide key insights into its regulation.

The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation: LARGE 3-SUBSTITUENTS PREVENT TAUTOMERIZATION

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H>F>Cl>Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A.3-Cl HOPDA and S112A.3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A.HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A.3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.

Formation and Reorientation of Glucose 1,6-Bisphosphate in the PMM/PGM Reaction: Transient-State Kinetic Studies

The interconversion of glucose 1-phosphate and glucose 6-phosphate, catalyzed by Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase, has been studied by transient-state kinetic techniques. Glucose 1,6-bisphosphate is formed as an intermediate in the reaction, but an obligatory step in the catalytic cycle appears to be the formation of an enzyme-glucose 1,6-bisphosphate complex that is not competent to form either glucose 1-phosphate or glucose 6-phosphate directly. We suggest that during the lifetime of this complex the glucose 1,6-bisphosphate intermediate undergoes the 180 degrees reorientation that is required for completion of the catalytic cycle. The formation of glucose 1,6-bisphosphate from glucose 1-phosphate is in rapid equilibrium relative to the rest of the reaction, where K(eq) = 0.14. In the opposite direction, glucose 1,6-bisphosphate is formed from glucose 6-phosphate with a rate constant of 12 s(-)(1), and the reverse step occurs with a rate constant of 255 s(-)(1). The interconversion of the productive and nonproductive glucose 1,6-bisphosphate complexes occurs with a rate constant of 64 s(-)(1) in one direction and 48 s(-)(1) in the other direction. Glucose 1,6-bisphosphate remains associated with the enzyme during reorientation. Isotope trapping studies indicate that it partitions to form glucose 1-phosphate or glucose 6-phosphate 14.3 times more frequently than it dissociates from the enzyme.

Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies

Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., “hot” peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 °C, pH 7.0). We determined rate constants approaching 2 × 10 7 and 5 × 10 5 M −1 s −1 for compound I and compound II reduction, respectively. We also determined k app values for steady-state capsaicinoid oxidation approaching 8 × 10 5 M −1 s −1 (25 °C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products.

The +2 NTP Binding Drives Open Complex Formation in T7 RNA Polymerase

Transcription initiation as catalyzed by T7 RNA polymerase consists primarily of promoter binding, strand separation, nucleotide binding, and synthesis of the first phosphodiester bond. The promoter strand separation process occurs at a very fast rate, but promoter opening is incomplete in the absence of the initiating NTPs. In this paper, we investigate how initiating NTPs affect the kinetics and thermodynamics of open complex formation. Transient state kinetic studies show that the open complex, ED(o), is formed via an intermediate ED(c), and the conversion of ED(c) to ED(o) occurs with an unfavorable equilibrium constant. In the presence of the initiating NTP that base-pairs with the template at position +2, the process of open complex formation is nearly complete. Our studies reveal that the nucleotide that drives open complex formation needs to be a triphosphate and to be correctly base-paired with the template. These results indicate that the melted template DNA in the open complex is positioned to bind the +2 NTP. The addition of +1 NTP alone does not stabilize the open complex; nor is it required for +2 NTP binding. However, there appears to be cooperativity in initiating NTP binding in that the binding of +2 NTP facilitates +1 NTP binding. The dissection of the initiation pathway provides insights into how open complex formation steps that are sensitive to the promoter sequence upstream from the initiation start site modulate the affinity of initiating NTPs and allow transcription initiation to be regulated by initiating NTP concentration.

Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding: HYDROGEN-DEUTERIUM EXCHANGE STUDIES USING MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY DETECTION

Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A. (1999) Biochemistry 38, 12072-12079). To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution.

Catalase-Peroxidases in Cyanobacteria – Similarities and Differences to Ascorbate Peroxidases

Cyanobacteria (blue-green algae) are oxygenic phototrophic bacteria carrying out plant-type photosynthesis. The only hydrogen peroxide scavenging enzymes in at least two unicellular species have been demonstrated to be bifunctional cytosolic catalase-peroxidases (CatPXs) having considerable homology at the active site with plant ascorbate peroxidases (APXs). In this paper we examined optical and kinetic properties of CatPXs from the cyanobacteria Anacystis nidulans and Synechocystis PCC 6803 and discuss similarities and differences to plant APXs. Both CatPXs and APX showed similar spectra of the ferric enzyme, the redox intermediate Compound I and the cyanide complex, whereas the spectrum of CatPX Compound II had characteristics reminiscent of the spectrum of the native enzyme. Both steady-state and multi-mixing transient-state kinetic studies were performed in order to characterize the kinetic behaviour of CatPXs. Bimolecular rate constants of both formation and reduction of a CatPX Compound I are presented. Because of its intrinsic high catalase activity (which cannot be found in APXs), the rate constants for Compound I formation were measured with peroxoacetic acid and are shown to be 5.9 x 10(4) M(-1) s(-1) for CatPX from A. nidulans and 8.7 x 10(3) M(-1) s(-1) for the Synechocystis enzyme. Compared with o-dianisidine (2.7-6.7 x 10(6) M(-1) s(-1)) and pyrogallol (8.6 x 10(4)-1.6 x 10(5) M(-1) s(-1)), the rate constant for Compound I reduction by ascorbate was extremely low (5.4 x 10(3) M(-1) s(-1) at pH 7.0 and 15 degrees C), in marked contrast to the behaviour of APXs.

Purification, Characterization, and Kinetic Studies of a SolubleBacteroides fragilis Metallo-β-lactamase That Provides Multiple Antibiotic Resistance

Resistance to multiple beta-lactam antibiotics traced to the expression of Zn(II) requiring metallo-beta-lactamases has emerged in clinical isolates of several bacterial strains including Bacteroides fragilis, a pathogen commonly found in suppurative/surgical infections. A soluble B. fragilis metallo-beta-lactamase has been purified to homogeneity from the cell growth medium after expression as a secretory protein in Escherichia coli. The enzyme requires two tightly bound Zn(II) ions for full activity, and the Zn(II) ions can be removed by EDTA from the enzyme. The apoenzyme is reactivated by stoichiometric amounts of Zn(II) and Co(II) ions. The Co(II)-substituted enzyme exhibits a UV-visible spectrum characterized by strong Co(II) d-d transitions at 510, 548, 615, and 635 nm and an EPR spectrum with g values of 5. 52, 4.25, and 2.01: features that serve as useful spectroscopic handles for the mechanistic studies of the enzyme. Although steady-state and transient-state kinetic studies of the soluble Zn(II) enzyme with nitrocefin as substrate found no ionizable groups with pKa values between 5.25 and 10.0 involved in catalysis, a kinetically significant proton transfer step in turnover was implicated by studies in deuterium oxide. These studies also detected the accumulation of an enzyme-bound intermediate and provide the basis for a minimal kinetic scheme describing metallo-beta-lactamase-catalyzed nitrocefin hydrolysis.

Haloperoxidase Activity of Manganese Peroxidase fromPhanerochaete chrysosporium

Manganese peroxidase (MnP) fromPhanerochaete chrysosporiumexhibits haloperoxidase activity at low pH. In the presence of hydrogen peroxide, MnP oxidizes bromide and iodide as measured by the formation of tribromide and triiodide complexes and the halogenation of various organic substrates. The optimum pHs for bromide and iodide oxidation are 2.5 and 3.0, respectively. Transient-state kinetic studies show that the reaction between MnP compound I and bromide or iodide occurs via a single two-electron step process, obeying second-order kinetics. The second-order rate constants for MnP compound I reduction by bromide and iodide are (4.1 ± 0.2) × 103and (1.1 ± 0.1) × 105m−1s−1, respectively, at pH 3.0. MnP brominates a variety of aromatic substrates, including veratryl (3,4-di-methoxybenzyl) alcohol (I) to produce of 2-bromo-4, 5-dimethoxybenzyl alcohol (II). MnP also hydrobrominates cinnamic acid (VI) to produce 2-bromo-3-hydroxyphenylpropionic acid (VII). With 3,4-dimethoxycinnamic acid (III) as the substrate, two bromination products are identified:trans-2-bromo-1-(3,4-dimethoxyphenyl) ethylene (IV) and 2-bromo-3-(3,4-dimethoxyphenyl)-3-hydroxypropionic acid (V). MnP also brominates 1,3-dicarbonyl compounds such as monochlorodimedone and malonic acid. Incubation of MnP with bromide and H2O2in the absence of organic substrates results in enzyme inactivation. MnP binds halides to produce characteristic optical difference spectra. From these spectra, apparent dissociation constants at pH 3.0 are determined to be 0.13, 20, and 45 mmfor fluoride, chloride, and bromide, respectively.

Lignin peroxidases can also oxidize manganese

The peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium include lignin peroxidases and manganese-dependent peroxidases. The major isozymes, called lignin peroxidases, are thought to oxidize chemicals directly. The manganese-dependent peroxidases (H3, H4, H5, and H9) are relatively minor, making up only a fraction of the total peroxidase protein. However, we have found that lignin peroxidases will also catalyze the H2O2-dependent oxidation of Mn2+ to Mn3+. We have used lignin peroxidase isozyme H2 (LiPH2) to characterize the manganese peroxidase activity of lignin peroxidases. Transient state kinetic studies were used to obtain a second-order rate constant of 4.2 x 10(4) M-1 S-1 for the reaction of LiPH2-compound I with free or chelated Mn2+ at pH 6.0. This reaction was too fast to monitor at pH 4.5. Only chelated Mn2+ could reduce LiPH2-compound II to ferric enzyme. The Mn(2+)-chelate (oxalate) first bound LiPH2-compound II with a Kd of (1.5 +/- 0.3) x 10(-5) M and then reduced LiPH2-compound II to ferric enzyme with a first order rate constant of 215 +/- 6 S-1. Steady-state kinetic studies on LiPH2 were performed by directly monitoring the formation of Mn(3+)-oxalate. These results show that oxidation of Mn2+ by a lignin peroxidase does not occur through free radical mediation as proposed previously [Popp et al. (1990) Biochemistry 29, 10475-10480). Electron spin resonance and oxygen evolution studies also indicate that Mn2+ is directly oxidized by LiPH2.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for Veratryl Alcohol as a Redox Mediator in Lignin Peroxidase-Catalyzed Oxidation

We have examined the hypothesis that veratryl alcohol (VA) may act as a redox mediator in lignin peroxidase (LiP)-catalyzed oxidations. The oxidation of chlorpromazine (CPZ) by this system was used to evaluate this hypothesis. Chlorpromazine can be oxidized by one electron to form a stable cation radical (CPZ.+). This cation radical can be oxidized by another electron to the sulfoxide (CPZSO). These oxidation steps are easily monitored, making CPZ a useful chemical to investigate redox mediation by VA. Lignin peroxidase oxidized CPZ to CPZ.+ whether or not VA was present. The inclusion of VA, however, stimulated CPZ oxidation to CPZ.+ and subsequent oxidation of CPZ.+ to CPZSO. In the absence of VA, the initial rates of CPZ oxidation by LiP were CPZ concentration-dependent. However, when saturating concentrations of VA were added, the oxidation of CPZ and CPZ.+ became independent of CPZ concentration. When the oxidation of VA to veratryl aldehyde was examined, increasing concentrations of CPZ produced a lag in veratryl aldehyde appearance proportional to the concentration of CPZ. Conversely, increasing concentrations of VA never inhibited CPZ oxidation. Transient-state kinetic studies indicated that both VA and CPZ reduced the compound I and compound II forms of LiP. However, when saturating concentrations of VA were utilized, LiP turnover was independent of CPZ concentration. We suggest these data demonstrate that VA may act as a redox mediator for the indirect oxidation of compounds by LiP.

Chemical and transient state kinetic studies on the formation and decomposition of horseradish peroxidase compounds XI and XII.

Previous studies on the chlorination reaction catalyzed by horseradish peroxidase using chlorite as the source of chlorine detected the formation of a chlorinating intermediate that was termed Compound X (Shahangian, S., and Hager, L.P. (1982) J. Biol. Chem. 257, 11529-11533). These studies indicated that at pH 10.7, the optical absorption spectrum of Compound X was similar to the spectrum of horseradish peroxidase Compound II. Compound X was shown to be quite stable at alkaline pH values. This study was undertaken to examine the relationship between the oxidation state of the iron protoporphyrin IX heme prosthetic group in Compound X and the chemistry of the halogenating intermediate. The experimental results show that the optical absorption properties and the oxidation state of the heme prosthetic group in horseradish peroxidase are not directly related to the presence of the activated chlorine atom in the intermediate. The oxyferryl porphyrin heme group in alkaline Compound X can be reduced to a ferric heme species that still retains the activated chlorine atom. Furthermore, the reaction of chlorite with horseradish peroxidase at acidic pH leads to the secondary formation of a green intermediate that has the spectral properties of horseradish peroxidase Compound I (Theorell, H. (1941) Enzymologia 10, 250-252). The green intermediate also retains the activated chlorine atom. By analogy to peroxidase Compound I chemistry, the heme prosthetic group in the green chlorinating intermediate must be an oxyferryl porphyrin pi-cation radical species (Roberts, J. E., Hoffman, B. M., Rutter, R. J., and Hager, L. P. (1981) J. Am. Chem. Soc. 103, 7654-7656). To be consistent with traditional peroxidase nomenclature, the red alkaline form of Compound X has been renamed Compound XII, and the green acidic form has been named Compound XI. The transfer of chlorine from the chlorinating intermediate to an acceptor molecule follows an electrophilic (rather than a free radical) path. A mechanism for the reaction is proposed in which the activated chlorine atom is bonded to a heteroatom on an active-site amino acid side chain. Transient state kinetic studies show that the initial intermediate, Compound XII, is formed in a very fast reaction. The second-order rate constant for the formation of Compound XII is approximately 1.1 x 10(7) M-1 s-1. The rate of formation of Compound XII is strongly pH-dependent. At pH 9, the second-order rate constant for the formation of Compound XII drops to 1.5 M-1 s-1. At acidic pH values, Compound XII undergoes a spontaneous first-order decay to yield Compound XI.(ABSTRACT TRUNCATED AT 400 WORDS)

Lignin peroxidase of Phanerochaete chrysosporium. Evidence for an acidic ionization controlling activity

The active site amino acid residues of lignin peroxidase are homologous to those of other peroxidases; however, in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with H2O2 to form compound I (Andrawis, A., Johnson, K.A., and Tien, M. (1988) J. Biol. Chem. 263, 1195-1198). Chloride binding is used in the present study to investigate this reaction further. Chloride binds to lignin peroxidase at the same site as cyanide and hydrogen peroxide. This is indicated by the following. 1) Chloride competes with cyanide in binding to lignin peroxidase. 2) Chloride is a competitive inhibitor of lignin peroxidase with respect to H2O2. The inhibition constant (Ki) is equal to the dissociation constant (Kd) of chloride at all pH values studied. Chloride binding is pH dependent: chloride binds only to the protonated form of lignin peroxidase. Transient-state kinetic studies demonstrate that chloride inhibits lignin peroxidase compound I formation in a pH-dependent manner with maximum inhibition at low pH. An apparent pKa was calculated at each chloride concentration; the pKa increased as the chloride concentration increased. Extrapolation to zero chloride concentration allowed us to estimate the intrinsic pKa for the ionization in the lignin peroxidase active site. The results reported here provide evidence that an acidic ionizable group (pKa approximately 1) at the active site controls both lignin peroxidase compound I formation and chloride binding. We propose that the mechanism for lignin peroxidase compound I formation is similar to that of other peroxidases in that it requires the deprotonated form of an ionizable group near the active site.

Electron Transfer within the Cytochromec-CytochromecPeroxidase Complex: Dependence of the Transient-State and Steady-State Kinetics on Ionic Strength

Abstract Steady-State and transient-state kinetic studies of the cytochrome c peroxidase (CcP) catalyzed oxidation of ferrocytochrome c by hydrogen peroxide at pH 7.5 as a function on ionic strength have been carried out using both horse heart and yeast iso-1 cytochrome c. The ionic strength affects the apparent interaction between cytochrome c and the oxidized intermediates of CcP while the intracomplex electron transfer rates are essentially independent of ionic strength. The maximum enzyme turnover rates for the oxidation of yeast iso-1 and horse heart ferrocytochrome c are 780 ± 130 s−1 and 280 ± 40 s−1, respectively, independent of ionic strength at pH 7.5. The intramolecular electron transfer rates from bound ferrocytochrome c to the heme site in CcP compound I is about 1900 s−1 and 450 s−1 for yeast iso-1 and horse heart cytochrome c, respectively.

Proton/product time course ratios: a new approach to transient-state kinetic analysis.

The usefulness of the indicator dye method for the detection of transient enzyme-product intermediates has been very limited due to the near impossibility of resolving the apparent single exponential time courses resulting from sequences of steps linked by rather similar rate constants. We propose here a novel approach, the proton-product time course method, a procedure which can extract a great deal of the mechanistic information which remains buried in conventional proton release-time course measurements. The method involves nothing more than measuring the ratio, r, of the moles of H+ released to the moles of product formed as a function of time. We derive the theory relating this r function to mechanisms of varying complexity, explore the theoretical behavior of the function in various possible mechanistic situations, and employ the new approach in an experimental system. We demonstrate the fact that the proton/product time course ratio method can provide evidence of the existence of hidden steps in transient state kinetic studies, that it can determine accurate thermodynamic pK values of the intermediate complexes involved in those steps, and that it can produce time courses of individual intermediates which are obscure to conventional kinetic methods.

Steady-state and transient-state kinetic studies on the oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerocheate chrysosporium Burds.

Catalysis of the H2O2-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H2O2 consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H2O2 (KM = 29 microM) and veratryl alcohol (KM = 72 microM) at pH 3.5. The magnitude of the turnover number varies from 2 to 3 s-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H2O2 reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK alpha approximately 3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH approximately 2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H2O2, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H2O2; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H2O2 and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates.

Transient-state kinetic studies on the mechanism of furylacryloylphosphatase-coupled calcium ion transport with sarcoplasmic reticulum adenosine triphosphatase.

Transient-state kinetic studies on the mechanism of furylacryloylphosphatase-coupled calcium ion transport with sarcoplasmic reticulum adenosine triphosphatase.