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PubMed1978

A unified theory of nucleation-rate-limited DNA renaturation kinetics.

DNA renaturations under nucleation-rate-limiting conditions on simple DNA such as bacterial and bacteriophage DNA show significant deviation from ideal second-order kinetics when followed by optical density measurements at 260 nm. Ideal second-order kinetics yield linear plots when the data is plotted in the standard reciprocal second-order (RSO) manner. The observed deviations from ideal second-order behavior take the form of steadily downward-curving RSO plots. In this paper, experiments are presented for E. coli and T2 DNA documenting this non-ideal behavior. Since experiments using T4, T5 and B, subtilis DNA yield identical non-ideal behavior, this behavior appears to be a property of DNA renaturation followed by optical density, not a peculiarity of a particular DNA. Identical non-ideal behavior is also seen in kinetics followed by S1 nuclease assay. A theory is developed to explain this deviation from ideal second-order kinetics. The theory also explains why kinetics followed by hydroxyapatite chromatography show nearly ideal second-order kinetics. In contrast to the approach taken by others in developing equations that describe S1 nuclease monitored reactions, our view is that nonideal second-order kinetics are fundamentally due to the reacton of free single strands to yield partially helical duplex species. Later reactions of these species tend to reduce the deviations from non-ideal second-order kinetics.

DNA↗

PubMed2005

Modelling atypical CYP3A4 kinetics: principles and pragmatism.

The Michaelis-Menten model, and the existence of a single active site for the interaction of substrate with drug metabolizing enzyme, adequately describes a substantial number of in vitro metabolite kinetic data sets for both clearance and inhibition determination. However, in an increasing number of cases (involving most notably, but not exclusively, CYP3A4), atypical kinetic features are observed, e.g., auto- and heteroactivation; partial, cooperative, and substrate inhibition; concentration-dependent effector responses (activation/inhibition); limited substrate substitution and inhibitory reciprocity necessitating sub-group classification. The phenomena listed above cannot be readily interpreted using single active site models and the literature indicates that three types of approaches have been adopted. First the 'nai ve' approach of using the Michaelis-Menten model regardless of the kinetic behaviour, second the 'empirical' approach (e.g., employing the Hill or uncompetitive inhibition equations to model homotropic phenomena of sigmoidicity and substrate inhibition, respectively) and finally, the 'mechanistic' approach. The later includes multisite kinetic models derived using the same rapid equilibrium/steady-state assumptions as the single-site model. These models indicate that 2 or 3 binding sites exist for a given CYP3A4 substrate and/or effector. Multisite kinetic models share common features, depending on the substrate kinetics and the nature of the effector response observed in vitro, which allow a generic model to be proposed. Thus although more complex than the other two approaches, they show more utility and can be comprehensively applied in relatively simple versions that can be readily generated from generic model. Multisite kinetic features, observed in isolated hepatocytes as well as in microsomes from hepatic tissue and heterologous expression systems, may be evident in substrate depletion-time profiles as well as in metabolite formation rates. Failure to adequately account for multisite kinetic phenomena will compromise any attempts to predict human drug clearance and drug-drug interaction potential from in vitro data.

Animals↗

PubMed2004

Reduced kinetic models of facilitative transport.

In spite of the highly complex structural dynamics of globular proteins, the processes mediated by them can usually be described in terms of relatively simple kinetic diagrams. How do complex proteins, characterized by undergoing transitions among a possibly very large number of intermediate states, exhibit functional properties that can be interpreted in terms of kinetic diagrams consisting of only a small number of states? One possible way of explaining this apparent contradiction is that, under some conditions, a reduction of the actual complete kinetic diagram that describes all of the macromolecular states and transitions takes place. In this work, we contribute with a formal basis to this interpretation, by generalizing the procedure of diagram reduction to the case of multicyclic kinetic diagrams. As an example, we apply the procedure to a complex kinetic model of facilitative transport. We develop Monte Carlo simulations to obtain the kinetic parameters of the complex model and we compare them with the ones analytically obtained from the reduced model. We confirm that, under some conditions, the kinetic behavior of the complex transporter is indistinguishable from the one of a four-state simple carrier model, derived from the former by diagram reduction. Besides introducing some novel methodological aspects, this work further contributes to the idea that, under many physiological and experimental conditions, a reduction occurs of the complete kinetic diagram that describes the dynamics of a globular protein.

Carrier Proteins↗

PubMed2006

Influence of functional bracing on the kinetics of anterior cruciate ligament-injured knees during level walking.

BACKGROUND: Use of functional knee braces has been suggested to provide protection and to improve kinetic performance of the knee in anterior cruciate ligament-injured patients. However, the efficacy of knee bracing in achieving these goals is still controversial. The purpose of this study was to examine the immediate effects of functional bracing on the three-dimensional kinetics of the knee in individuals with anterior cruciate ligament injuries during level walking. METHODS: Fifteen anterior cruciate ligament-deficient and 15 anterior cruciate ligament-reconstructed subjects were each fitted with a DonJoy Goldpoint brace and walked at a self-selected pace, first without and then with the brace. Kinematic and kinetic data were measured and three-dimensional joint moments and angular impulses at the knee were calculated and compared between bracing conditions and between limbs. FINDINGS: Functional knee bracing did not significantly affect the kinetics of the unaffected knees for either group. Bracing significantly increased the peak abductor moments in anterior cruciate ligament-deficient knees and reduced the bilateral kinetic asymmetry in the coronal plane. For the anterior cruciate ligament-reconstructed group, bracing increased peak moments and impulses of the abductors and extensors. It also reduced bilateral kinetic asymmetry in the sagittal and coronal planes. INTERPRETATION: Effects of the knee brace were apparent in the coronal plane for both anterior cruciate ligament-deficient and anterior cruciate ligament-reconstructed patients, and in the sagittal plane for anterior cruciate ligament-reconstructed patients. Functional bracing can be recommended for anterior cruciate ligament-reconstructed patients to assist in achieving better bilateral kinetic symmetry during gait. For anterior cruciate ligament-deficient patients, apart from bracing, additional emphasis on the rehabilitative training for better kinetic knee performance in the sagittal plane is needed.

Adult↗

PubMed2006

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis.

Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward- and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in EX1 kinetic events.

Amides↗

PubMed2006

Role of solvation barriers in protein kinetic stability.

The stability of several protein systems of interest has been shown to have a kinetic basis. Besides the obvious biotechnological implications, the general interest of understanding protein kinetic stability is emphasized by the fact that some emerging molecular approaches to the inhibition of amyloidogenesis focus on the increase of the kinetic stability of protein native states. Lipases are among the most important industrial enzymes. Here, we have studied the thermal denaturation of the wild-type form, four single-mutant variants and two highly stable, multiple-mutant variants of lipase from Thermomyces lanuginosa. In all cases, thermal denaturation was irreversible, kinetically controlled and conformed to the two-state irreversible model. This result supports that the novel molecular-dynamics-focused, directed-evolution approach involved in the preparation of the highly stable variants is successful likely because it addresses kinetic stability and, in particular, because heated molecular dynamics simulations possibly identify regions of disrupted native interactions in the transition state for irreversible denaturation. Furthermore, we find very large mutation effects on activation enthalpy and entropy, which were not accompanied by similarly large changes in kinetic urea m-value. From this we are led to conclude that these mutation effects are associated to some structural feature of the transition state for the irreversible denaturation process that is not linked to large changes in solvent accessibility. Recent computational studies have suggested the existence of solvation/desolvation barriers in at least some protein folding/unfolding processes. We thus propose that a solvation barrier (arising from the asynchrony between breaking of internal contacts and water penetration) may contribute to the kinetic stability of lipase from T. lanuginosa (and, possibly, to the kinetic stability of other proteins as well).

Ascomycota↗

PubMed2006

Measurement of kinetically resolved vesicular dopamine uptake and efflux using rotating disk electrode voltammetry.

The vesicular monoamine transporter-2 (VMAT-2) sequesters cytoplasmic dopamine (DA) into vesicles for storage and subsequent release. VMAT-2 activity has traditionally been measured in small synaptic vesicles isolated from rat striatum by monitoring [3H] DA uptake and in cellular expression systems using fast scan cyclic voltammetry. This is the first report using rotating disk electrode (RDE) voltammetry to measure VMAT-2 DA uptake and efflux in small synaptic vesicles. DA uptake profiles followed mixed order kinetics with apparent zero order kinetics for the first 25 s and apparent first order kinetics thereafter. Vesicular DA uptake was temperature- and ATP-dependent and was blocked by the VMAT-2 inhibitor tetrabenazine. Initial velocities of DA uptake were kinetically resolved and displayed Michaelis-Menten kinetics with a Km and Vmax of 289 +/- 59 nM and 1.9 +/- 0.2 fmol/(s microg protein), respectively. Methamphetamine-induced DA efflux was blocked by tetrabenazine and kinetically resolved with an initial velocity of 0.54 +/- 0.08 fmol/(s microg protein). These results suggest that RDE voltammetry can be used to make kinetically resolved measurements of vesicular DA uptake and efflux and will allow the design of experiments that could reveal important information about the kinetics of VMAT-2 activity and its inhibition.

Adenosine Triphosphate↗

PubMed1996

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.

Animals↗

PubMed1997

Protein folding kinetics: timescales, pathways and energy landscapes in terms of sequence-dependent properties.

BACKGROUND: Recent experimental and theoretical studies have revealed that protein folding kinetics can be quite complex and diverse depending on various factors such as size of the protein sequence and external conditions. For example, some proteins fold apparently in a kinetically two-state manner, whereas others follow complex routes to the native state. We have set out to provide a theoretical basis for understanding the diverse behavior seen in the refolding kinetics of proteins in terms of properties that are intrinsic to the sequence. RESULTS: The folding kinetics of a number of sequences for off-lattice continuum models of proteins is studied using Langevin simulations at two different values of the friction coefficient. We show for these models that there is a remarkable correlation between folding time, tau F, and sigma = (T theta - TF)/T theta, where T theta and TF are the equilibrium collapse and folding transition temperatures, respectively. The microscopic dynamics reveals that several scenarios for the kinetics of refolding arise depending on the range of values of sigma. For relatively small sigma, the chain reaches the native conformation by a direct native conformation nucleation collapse (NCNC) mechanism without being trapped in any detectable intermediates. For moderate and large values of sigma, the kinetics is described by the kinetic partitioning mechanism, according to which a fraction of molecules phi (kinetic partition factor) reach the native conformation via the NCNC mechanism. The remaining fraction attains the native state by off-pathway processes that involve trapping in several misfolded structures. The rate-determining step in the off-pathway processes is the transition from the misfolded structures to the native state. The partition factor phi is also determined by sigma: the smaller the value of sigma, the larger is phi. The qualitative aspects of our results are found to be independent of the friction coefficient. The simulation results and theoretical arguments are used to obtain estimates for timescales for folding via the NCNC mechanism in small proteins, those with less than about 70 amino acid residues. CONCLUSIONS: We have shown that the various scenarios for folding of proteins, and possibly other biomolecules, can be classified solely in terms of sigma. Proteins with small values of sigma reach the native conformation via a nucleation collapse mechanism and their energy landscape is characterized by having one dominant native basin of attraction (NBA). On the other hand, proteins with large sigma get trapped in competing basins of attraction (CBAs) in which they adopt misfolded structures. Only a small fraction of molecules access the native state rapidly when sigma is large. For these sequences, the majority of the molecules approach the native state by a three-stage multipathway mechanism in which the rate-determining step involves a transition from one of the CBAs to the NBA.

Kinetics↗

PubMed2000

Effects of regulatory subunits on the kinetics of protein phosphatase 2A.

Both the scaffold (A) and the regulatory (R) subunits of protein phosphatase 2A regulate enzyme activity and specificity. Heterotrimeric enzymes containing different R-subunits differ in their specific activities for substrates. Kinetic parameters for the dephosphorylation of a phosphopeptide by different oligomeric forms of PP2A were determined to begin to elucidate the molecular basis of regulatory subunit effects on phosphatase activity. Using steady state kinetics and the pH dependence of kinetic parameters, we have explored the effect of the A- and R-subunits on the kinetic and chemical mechanism of PP2A. The regulatory subunits affected a broad range of kinetic parameters. The C-subunit and AC dimer were qualitatively similar with respect to the product inhibition patterns and the pH dependence of kinetic parameters. However, a 22-fold decrease in rate and a 4.7-fold decrease in K(m) can be attributed to the presence of the A-subunit. The presence of the R2alpha (Balpha or PR55alpha) subunit caused an additional decrease in K(m) and changed the kinetic mechanism of peptide dephosphorylation. The R2alpha-subunit also caused significant changes in the pH dependence of kinetic parameters as compared to the free C subunit or AC heterodimer. The data support an important role for the regulatory subunits in determining both the affinity of PP2A heterotrimers for peptide substrates and the mechanism by which they are dephosphorylated.

Amino Acid Sequence↗

PubMed1984

Transient kinetics of the binding of ATP to actomyosin subfragment 1: evidence that the dissociation of actomyosin subfragment 1 by ATP leads to a new conformation of subfragment 1.

The initial steps by which ATP dissociates and binds to actomyosin subfragment 1 (acto-SF-1) were studied. Two techniques were used: stopped-flow (for acto-SF-1 dissociation kinetics) and rapid-flow quench with ATP chase quenching (for ATP binding kinetics). The experiments were carried out in 40% ethylene glycol-5 mM KCI, pH 8, at 15 degrees C. Under these conditions, the binding of SF-1 to actin remains very tight. As with SF-1, the ATP chase technique could be used, first, to titrate active sites and, second, to study the kinetics of ATP binding to acto-SF-1. The kinetic constants obtained were compared with those of SF-1 alone and with the acto-SF-1 dissociation kinetics under identical conditions. The kinetics of the acto-SF-1 dissociation did not vary with the actin to SF-1 ratio, but the ATP binding kinetics did, and a maximum value was reached at a mole ratio of 2.5. At high ATP (100 microM), kdiss = 300 s-1, which compares with 49 s-1 and 13 s-1 for the ATP binding kinetics for acto-SF-1 (actin to SF-1 = 1:1) and SF-1, respectively. As with SF-1, the ATP binding to acto-SF-1 follows a hyperbolic law with the ATP concentration. This suggests a rapid equilibrium (K) followed by an essentially irreversible step (k).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed2004

Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward beta-sheet structure.

The term kinetic stability is used to describe proteins that are trapped in a specific conformation because of an unusually high-unfolding barrier that results in very slow unfolding rates. Motivated by the observation that some proteins are resistant to sodium dodecyl sulfate (SDS)-induced denaturation, an attempt was made to determine whether this property is a result of kinetic stability. We studied many proteins, including a few kinetically stable proteins known to be resistant to SDS. The resistance to SDS-induced denaturation was investigated by comparing the migration on polyacrylamide gels of identical boiled and unboiled protein samples containing SDS. On the basis of the different migration of these samples, eight proteins emerged as being resistant to SDS. The kinetic stability of these proteins was confirmed by their slow unfolding rate upon incubation in guanidine hydrochloride. Further studies showed that these proteins were also extremely resistant to proteolysis by proteinase K, suggesting that a common mechanism may account for their resistance to SDS and proteolytic cleavage. Together, these observations suggest that a rigid protein structure may be the physical basis for kinetic stability and that resistance to SDS may serve as a simple assay for identifying proteins whose native conformations are kinetically trapped. Remarkably, most of the kinetically stable SDS-resistant proteins in this study are oligomeric beta-sheet proteins, suggesting a bias of these types of structures toward kinetic stability.

Avidin↗

PubMed2006

In situ kinetics of cytochromes c1 and c2.

In Rhodobacter sphaeroides chromatophores, cytochromes (cyt) c(1) and c(2) have closely overlapping spectra, and their spectral deconvolution provides a challenging task. As a result, analyses of the kinetics of different cytochrome components of the bc(1) complex in purple bacteria usually report only the sum cyt c(1) + cyt c(2) kinetics. Here we used newly determined difference spectra of individual components to resolve the kinetics of cyt c(1) and c(2) in situ via a least-squares (LS) deconvolution. We found that the kinetics of cyt c(1) and c(2) are significantly different from those measured using the traditional difference wavelength (DW) approach, based on the difference in the absorbance at two different wavelengths specific for each component. In particular, with the wavelength pairs previously recommended, differences in instrumental calibration led to kinetics of flash-induced cyt c(1) oxidation measured with the DW method which were faster than those determined by the LS method (half-time of approximately 120 micros vs half-time of approximately 235 micros, in the presence of antimycin). In addition, the LS approach revealed a delay of approximately 50 micros in the kinetics of cyt c(1) oxidation, which was masked when the DW approach was used. We attribute this delay to all processes leading to the oxidation of cyt c(1) after light activation of the photosynthetic reaction center, especially the dissociation of cyt c(2) from the reaction center. We also found that kinetics of both cyt c(1) and c(2) measured by the DW approach were significantly distorted at times longer than 1 ms, due to spectral contamination from changes in the b hemes. The successful spectral deconvolution of cyt c(1) and c(2), and inclusion of both cytochromes in the kinetic analysis, significantly increase the data available for mechanistic understanding of bc(1) turnover in situ.

Antimycin A↗

PubMed1996

Kinetics of nonenzymatic glycation of ribonuclease A leading to advanced glycation end products. Paradoxical inhibition by ribose leads to facile isolation of protein intermediate for rapid post-Amadori studies.

Nonenzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes and possibly aging and Alzheimer's disease. We report here kinetic studies of the glycation of the model protein ribonuclease A by glucose and ribose leading to the formation of antigenic advanced glycation end products ("AGEs"), detectable by AGE-specific polyclonal antibodies, and pentosidine, an acid-stable fluorescent AGE. As anticipated, the kinetics of glycation by ribose were considerably faster than by glucose, and the rate of AGE formation initially increased with increasing sugar concentrations. However, ribose above 0.15 M appeared to paradoxically slow the kinetics of AGE formation, suggesting ribose inhibits the conversion of "early" Amadori rearrangement products to "late" AGEs and thus favors the accumulation of reactive Amadori intermediates. The facile isolation of such protein intermediates was achieved by an "interrupted glycation" protocol which free and reversibly bound (Schiff base) ribose was removed following a short (24h) initial incubation of 0.5 M ribose at 37 degrees C. The kinetics of buildup of the Amadori intermediates and the kinetics of their post-Amadori conversion to antigenic AGEs were independently studied. A rapid and reversible inhibition of the post-Amadori kinetics by free ribose was verified by direct re-addition of ribose to the isolated, sugar-free intermediate. The pH dependence of the kinetics of antigenic AGE formation from such intermediates was measured and exhibited an unusual bell-shaped profile over the pH range of 5.0-9.5 with a maximum near pH 8.0. Aminoguanidine, a pharmacological AGE inhibitor, was found to moderately or weakly inhibit antigenic AGE formation in such post- Amadori steps. The isolation of the glycated ribonuclease intermediate thus simplifies kinetic and mechanistic studies of AGE formation, permits AGE studies in the absence of complications arising from free or Schiff base bound sugar, and provides a novel methodology for evaluating the mechanism and efficacy of therapeutic agents that may inhibit AGE formation.

Antibodies↗

PubMedDate not supplied

Investigations of reaction kinetics for immobilized enzymes--identification of parameters in the presence of diffusion limitation.

A method is proposed for identification of kinetic parameters when diffusion of substrates is limiting in reactions catalyzed by immobilized enzymes. This method overcomes conventional sequential procedures, which assume immobilization does not affect the conformation of the enzyme and, thus, consider intrinsic and inherent kinetics to be the same. The coupled equations describing intraparticle mass transport are solved simultaneously using numerical methods and are used for direct estimation of kinetic parameters by fitting modeling results to time-course measurements in a stirred tank reactor. While most traditional procedures were based on Michaelis-Menten kinetics, the method presented here is applicable to more complex kinetic mechanisms involving multiple state variables, such as ping-pong bi-bi. The method is applied to the kinetic resolution of (R/S)-1-methoxy-2-propanol with vinyl acetate catalyzed by Candida antarctica lipase B. A mathematical model is developed consisting of irreversible ping-pong bi-bi kinetics, including competitive inhibition of both enantiomers. The kinetic model, which fits to experimental data over a wide range of both substrates (5-95%) and temperatures (5-56 degrees C), is used for simulations to study typical behavior of immobilized enzyme systems.

Biotechnology↗

PubMed2002

Kinetics and steady-state of VO2 responses to arm exercise in trained spinal cord injury humans.

STUDY DESIGN: Cross-sectional study comparing trained spinal cord injured (SCI) subjects (lesion level: L1 - T6) with healthy young subjects (CONT). OBJECTIVE: To investigate the kinetics of response in oxygen uptake (VO(2)) in human upper-body skeletal muscles, nine trained SCI subjects underwent submaximal supine arm exercises. METHOD: The SCI subjects underwent an incremental arm exercise test until exhaustion. The days after this first round of testing, breath-by-breath VO(2) and beat-by-beat heart rate (HR) on- and off-kinetics were determined during three repetitions of constant exercise at 50% of VO(2peak). The overall time course of response was determined from the half time (t(1/2)). Increased capillary blood lactate production (delta[La]b) at the onset of exercise was defined as the difference between at rest and at the end of exercise. Cardiac output (Q) was measured using the acetylene rebreathing method during the steady state of exercise. In accordance with the Fick principle, the difference in arterial-venous O(2) content (Ca-vO(2)) was defined as VO(2)/Q. RESULTS: During the steady state of the submaximal arm exercise, a more significant increase in the steady state of Q was obtained in the CONT subjects than in the trained SCI subjects: respectively, 14.9+/-1.4 l/min versus (12.7+/-0.8 l/min). There was no difference in the steady state of VO(2) between the two groups; as a result, SCI subjects had the greater Ca-v(2). Meanwhile, VO(2) on- and off-kinetics became much faster in the trained SCI subjects than in the CONT subjects. In addition, t(1/2) HR on-kinetics was not significantly different between the SCI and CONT groups. Increased Delta[La]b was closely related to larger t(1/2) VO(2) on-kinetics (r = 0.624, P < 0.05). CONCLUSION: It is concluded that the acceleration of VO(2) on- and off-kinetics in the trained SCI subjects was observed even though there was no difference in HR on- and off-kinetics between the SCI and CONT groups and a lower steady state of Q in the trained SCI subjects. VO(2) kinetics would therefore be the limiting factor in oxidative phosphorylation in the upper skeletal muscles, thereby providing a lower lactic O(2)-deficit (ie delta[La]b).

Adult↗

PubMed1993

Clarification of the pH-dependent kinetic behaviour of papain by using reactivity probes and analysis of alkylation and catalysed acylation reactions in terms of multihydronic state models: implications for electrostatics calculations and interpretation of the consequences of site-specific mutations such as Asp-158-Asn and Asp-158-Glu.

1. The complex behaviour of papain (EC 3.4.22.2) in acidic media has been investigated by (a) stopped-flow reactivity probe kinetics using 4,4'-dipyrimidyl disulphide (I) and 2,2'-dipyridyl disulphide (II) as thiol-specific time-dependent inhibitors with markedly different susceptibilities to activation by hydronation (protonation) and (b) using the multitasking application program SKETCHER for the rapid evaluation of pH-dependent kinetic data by means of interactive manipulation of calculated curves. 2. The substantially lower basicity of (I) (pKa 0.91) than that of (II) (pKa 2.45) combined with retention of high reactivity permitted the pKa for the formation of the (Cys-25)-S-/(His-159)-Im+H ion-pair state of papain to be determined kinetically as 3.4, a value close to that (3.3) deduced by potentiometric difference titration [Lewis, Johnson and Shafer (1976) Biochemistry 15, 5009-5017] and lower than the value (approx. 4) often reported from pH-dependent kinetic studies. The higher values are now known to arise from inadequate data analysis that does not take account of other overlapping kinetically influential ionizations. 3. Re-evaluation of the extensive sets of pH-kcat/Km data for the hydrolysis of nine substrates by papain reported by Polgár and Halász (1978) (Eur. J. Biochem. 88, 513-521) by making use of SKETCHER, the known pKa value (3.4) from the reaction with compound (I) and two additional kinetically influential pKa values deduced from the reaction with compound (II) now permits the identification of the pH-dependent events in reactions of papain with inhibitors and substrates. 4. A major conclusion is that, whereas in reactions of simple alkylating agents and compound (I) full nucleophilic character of (Cys-25)-S-/(His-159)-Im+H is provided by hydronic dissociation with pKa 3.3-3.4, in catalysis relatively little catalytic competence is produced consequent upon ion-pair formation. Substantial catalytic competence requires further hydronic dissociation with pKa approx. 4, and for cationic substrates further enhancement is produced by hydronic dissociation with pKa approx. 5. 5. The present work, together with the kinetic analysis of reactions of papain in alkaline media reported by Mellor, Thomas, Topham and Brocklehurst [Biochem. J. (1993) 290, 289-296], defines the kinetically influential ionizations of papain as 3.4, 4.0, 5.0, 8.3 and 10.0 of which 3.4 and 8.3 relate to the formation and subsequent dehydronation of the ion-pair state.(ABSTRACT TRUNCATED AT 400 WORDS)

Acylation↗

PubMed1979

Determination of enzyme mechanisms by radiationless energy transfer kinetics.

Rigorous definition of the elementary steps of an enzymatic reaction requires visualization of transient enzyme-substrate (ES) complexes. Measurement of radiationless energy transfer (RET) between enzyme tryptophan residues and a fluorescent dansyl (5-dimethylaminonaphthalene-1-sulfonyl) substrate provides a sensitive means to observe ES complexes directly. Analysis of the rate of formation and breakdown of ES complexes by RET can serve as the basis of a rapid kinetic approach to enzyme mechanisms. Both pre-steady-state and steady-state kinetics can be performed in the same RET experiment. Analysis at steady state precisely determines k(cat) and K(m) values by multiple means. Analysis at pre-steady state determines the number of intermediates, the type of reaction mechanism, and all the individual binding and rate constants. Chymotrypsin was chosen as a standard of reference for RET kinetics because extensive investigations have established both the existence of transient intermediates in the course of its catalytic process and the range of values to be expected for pertinent kinetic constants. As predicted, RET kinetics readily detects the two known intermediates in the alpha-chymotrypsincatalyzed hydrolysis of specific ester substrates. The results are both qualitatively and quantitatively in accord with data derived for this enzyme from classical kinetics. Hence, this experimental study both validates and demonstrates the theoretical advantages and potential of RET kinetics. The generality of the approach has been investigated by synthesizing a family of dansyl-labeled substrates designed to meet the specificity requirements of a number of metallo- and nonmetallo- exo- and endopeptidases. In all cases, the ES complex is observed readily at micromolar or lower concentrations of enzyme under stopped-flow conditions. The success of the RET kinetic approach on proteolytic enzymes shows its broad utility.

Carboxypeptidases↗