Rapid Reaction Techniques Research Articles

Professor H. Gutfreund, FRS (21 October 1921 to 21 March 2021)

Professor H. Gutfreund, FRS (21 October 1921 to 21 March 2021)

Steam and Flame Applications as Novel Methods of Population Control for Invasive Asian Clam (Corbicula fluminea) and Zebra Mussel (Dreissena polymorpha)

Control strategies for established populations of invasive alien species can be costly and complex endeavours, which are frequently unsuccessful. Therefore, rapid-reaction techniques that are capable of maximising efficacy whilst minimising environmental damage are urgently required. The Asian clam (Corbicula fluminea Müller, 1774), and the zebra mussel (Dreissena polymorpha Pallas, 1771), are invaders capable of adversely affecting the functioning and biodiversity of freshwater ecosystems. Despite efforts to implement substantial population-control measures, both species continue to spread and persist within freshwater environments. As bivalve beds often become exposed during low-water conditions, this study examined the efficacy of steam-spray (≥100 °C, 350 kPa) and open-flame burn treatments (~1000 °C) to kill exposed individuals. Direct steam exposure lasting for 5 min caused 100% mortality of C. fluminea buried at a depth of 3 cm. Further, combined rake and thermal shock treatments, whereby the substrate is disturbed between each application of either a steam or open flame, caused 100% mortality of C. fluminea specimens residing within a 4-cm deep substrate patch, following three consecutive treatment applications. However, deeper 8-cm patches and water-saturated substrate reduced maximum bivalve species mortality rates to 77% and 70%, respectively. Finally, 100% of D. polymorpha specimens were killed following exposure to steam and open-flame treatments lasting for 30 s and 5 s, respectively. Overall, our results confirm the efficacy of thermal shock treatments as a potential tool for substantial control of low-water-exposed bivalves. Although promising, our results require validation through upscaling to field application, with consideration of other substrate types, increased substrate depth, greater bivalve densities, non-target and long-term treatment effects.

Fluorescence to study the ATPase mechanism of motor proteins.

This chapter provides an overview of different methodologies to dissect the ATPase mechanism of motor proteins. The use of ATP is fundamental to how these molecular engines work and how they can use the energy to perform various cellular roles. Rapid reaction and single-molecule techniques will be discussed to monitor reactions in real time through the application of fluorescence intensity, anisotropy and FRET. These approaches utilise fluorescent nucleotides and biosensors. While not every technique may be suitable for your motor protein, the different ways to determine the ATPase mechanism should allow a good evaluation of the kinetic parameters.

Mechanism of Potentiation of a Dysfunctional Epilepsy-Linked Mutated GABAA Receptor by a Neurosteroid (3α, 21-Dihydroxy-5α-pregnan-20-one): Transient Kinetic Investigations

The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46-48] has been accounted for by a 5-fold decrease in the channel-opening equilibrium of the mutated receptor compared to the wild type [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. Here we describe the mechanism by which the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) alleviates this malfunction of the mutated receptor transiently expressed in HEK293 cells. Two rapid reaction techniques, the cell-flow and the laser-pulse photolysis methods, were used in combination with whole-cell current recordings. 150-muM 5alpha-THDOC does not affect the rate constant for channel opening (k(op)) of approximately 250 s(-1) but does decrease the rate constant for channel closing (k(cl)) from 121 +/- 11 s(-1) to 56 +/- 21 s(-1). This results in an increase in the channel-opening equilibrium constant ((Phi(-1) = k(op)/k(cl)) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (alpha1beta2gamma2L(K289M)) GABA(A) receptor linked to GEFS+.

Effect of Salt and pH on the Reductive Half-Reaction of Mycobacterium tuberculosis FprA with NADPH

Despite a number of studies, the formation of the Michaelis complexes between ferredoxin-NADP (+) reductases and NADP(H) eluded detailed investigations by rapid kinetic techniques because of their high formation rates. Moreover, the reversible nature of the reaction of hydride ion transfer between these enzymes and NADPH prevented the obtainment of reliable estimates of the rate constant of the hydride transfer step. Here we show that by working at a high salt concentration, the mechanism of the reaction with NADPH of FprA, a Mycobacterium tuberculosis homologue of adrenodoxin reductase, is greatly simplified, making it amenable to investigation by rapid reaction techniques. The approach presented herein allowed for the first time the observation of the formation of the Michaelis complex between an adrenodoxin reductase-like enzyme and NADPH, and the determination of the related rate constants for association and dissociation. Furthermore, the rate constant for the reaction of hydride ion transfer between NADPH and FAD could be unambiguously assessed. It is proposed that the approach described should be applicable to other ferredoxin reductase enzymes, providing a valuable experimental tool for the study of their kinetic properties.

Rapid chemical reaction techniques developed for use in investigations of membrane-bound proteins (neurotransmitter receptors)

New techniques for investigating chemical reactions on cell surfaces in the microsecond-to-millisecond time region are described. Reactions mediated by membrane-bound neurotransmitter receptors that control signal transmission between ∼1012 cells of the nervous system are taken as an example. Cells with receptors on their plasma membrane are equilibrated with photolabile, biologically inactive precursors of the neurotransmitters. Photolysis of these compounds releases free neurotransmitter that interacts with the receptors, leading to the transient opening of transmembrane receptor-formed channels that are permeant to small inorganic ions. The current thus induced can be measured. The technique can be used to measure the elementary steps of the receptor-mediated reactions. To illustrate the approach it was shown that an understanding of the mechanism of inhibition of the nicotinic acetylcholine receptor by the drug cocaine was obtained and led to the first proof that compounds exist that alleviate the inhibition.

Mechanism of glutamate receptor-channel function in rat hippocampal neurons investigated using the laser-pulse photolysis (LaPP) technique.

Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).

Synthesis and photochemistry of a photolabile precursor of N-methyl-D-aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor.

The amino acid L-glutamate is a major neurotransmitter at excitatory synapses within the central nervous system. Neuronal responses to glutamate are mediated by at least three receptor types, one of which is the NMDA subtype, named for its specific ligand N-methyl-D-aspartic acid. Neurotransmitter receptors are transmembrane proteins that can form ion channels upon binding a specific ligand and are involved in many physiological activities of the brain and in some neurological disorders. Elucidating the mechanisms of the formation of transmembrane receptor-channels and of receptor regulation and inhibition is necessary for understanding nervous system function and for designing potential therapeutic agents. This has been hampered by the lack of rapid reaction techniques suitable for investigating protein-mediated reactions on cell surfaces. Recently a laser-pulse photolysis technique was developed to study the chemical reactions of channel-forming receptor proteins in the microsecond-to-millisecond time region. To apply the technique to NMDA1 receptors a photolabile NMDA precursor (beta-DNB NMDA) was synthesized. In this precursor the side chain carboxylate was protected as a photosensitive 2,2'-dinitrobenzhydryl ester. Photolysis with 308 nm laser light generated free NMDA with a time constant of 4.2 +/- 0.1 microseconds at pH 7 and a photolysis quantum yield of 0.18 +/- 0.05. In rat hippocampal neurons the beta-DNB NMDA (250 microM) neither activated endogenously expressed receptors nor potentiated or inhibited the NMDA response. Equilibration of hippocampal neurons in the whole-cell current recording mode with 250 microM caged precursor followed by a pulse of 333 nm laser light resulted in a rapid current rise with a rate constant of 100 s-1 due to opening of NMDA-activated receptor-channels. The caged NMDA precursor described here now makes it possible to investigate the mechanism of NMDA receptors in the micro- to millisecond time region.

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes.

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-microseconds time resolution, respectively, for studying neurotransmitter receptors in giant (approximately 20-microns diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.

The transient kinetics of Escherichia coli chorismate synthase: substrate consumption, product formation, phosphate dissociation, and characterization of a flavin intermediate.

Chorismate synthase is the seventh enzyme of the shikimate pathway and catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The reaction involves the 1,4-elimination of phosphate and the C-(6proR) hydrogen of the substrate with unusual anti stereochemistry and requires a reduced flavin cofactor. This paper describes the kinetics of the formation and decay of a flavin intermediate, EPSP consumption, chorismate and phosphate formation, and phosphate dissociation during single and multiple turnover experiments, determined using rapid reaction techniques. The kinetics of phosphate dissociation using the substrate analogues (6R)-[6-2H]EPSP and (6S)-6-fluoro-EPSP have also been determined. The observations are consistent with a nonconcerted chorismate synthase reaction. The flavin intermediate is not simply associated with the conversion of substrate to product because it forms before the substrate is consumed. The transient spectral changes must be associated primarily with events such as protonation of the reduced flavin, a charge transfer complex between reduced flavin and an aromatic amino acid, or a conformational change in the protein. This does not rule out the direct role of flavin in catalysis.

Synapsis by Tn3 resolvase: speed and dependence on DNA supercoiling.

Synapsis by Tn3 resolvase: speed and dependence on DNA supercoiling.

Determination of the chemical mechanism of neurotransmitter receptor-mediated reactions by rapid chemical kinetic techniques.

Rapid chemical reaction techniques play an important role in unraveling the mechanism of reactions mediated by soluble proteins, including enzymes involved in the regulation of intracellular processes and the biosynthesis of proteins and nucleic acids. Regulatory proteins change conformation rapidly and must, therefore, be studied in the microsecond-to-millisecond time region. Similar techniques for investigating reactions mediated by membrane-bound neurotransmitter receptors were not available, and the mechanisms of the reactions are poorly understood. These proteins must be studied in a membrane-bound form in cells or vesicles, over a wide range of reactant concentrations, and in the microsecond-to-millisecond time region. Four rapid-mixing techniques for making kinetic measurements in the microsecond and millisecond time regions have now been developed for investigating neurotransmitter receptors in the membranes of neurons and muscle cells, thus extending chemical kinetic approaches to membrane-bound proteins and intercellular processes. Neurotransmitter receptors regulate transmission of signals between neurons (approximately 10(12) in the human nervous system), thereby allowing perception of stimuli, integration and storage of information, and reaction to the environment. Six structurally related neurotransmitter receptors, and many isoforms, have been identified by use of recombinant DNA technology. Modern electrophysiological techniques show that these different proteins, upon binding a specific chemical signal (neurotransmitter), transiently open transmembrane channels, which are characterized by their ion selectivity, conductance, and lifetime. To be able to account for the receptor-mediated voltage changes that trigger signal transmission between cells, we still need to know the concentration of open receptor-channels. This concentration changes with time and is affected by the concentration of neurotransmitter. Rapid reaction techniques are particularly suitable for determining the relationship between neurotransmitter concentration and the time-dependent concentration of the open receptor-channels. The four rapid reaction techniques adapted or developed for studying receptor mechanisms are quench- and stopped-flow, adapted for use with vesicles, and cell-flow and laser-pulse photolysis for use with single cells. The approach was initiated when it was found that the neurotransmitter receptors desensitize (become transiently inactive) faster, by almost two orders of magnitude, than was believed. Before fast reaction techniques were used, the chemical properties of only desensitized forms were investigated, although this was not recognized. So far, the chemical mechanism(s) of the excitatory (cation-specific) acetylcholine receptor in membrane vesicles, electroplax cells, and single clonal cells, and the inhibitory (anion-specific) gamma-aminobutyric acid (GABA) receptor in primary cerebral cortical cells have been investigated with the new techniques.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron-transfer steps involved in the reactivity of Hansenula anomala flavocytochrome b2 as deduced from deuterium isotope effects and simulation studies

The L-lactate-flavocytochrome b2-ferricyanide electron-transfer system from the yeast Hansenula anomala was investigated by rapid-reaction techniques. The kinetics of reduction of oxidized flavocytochrome b2 by L-lactate and L-[2H]lactate were biphasic both for flavin and haem prosthetic groups and at all concentrations tested. The first-order rate constants of the rapid and slow phases depended upon substrate concentrations, a saturation behaviour being exhibited. Substitution of the C alpha-H atom by 2H was found to cause appreciable changes in the rate constants for the initial reduction of flavin and haem (phase I), which were respectively about 3-fold and 2-fold less than with L-lactate. In contrast, no significant isotope effect was noted on the apparent reduction rate constants of the slow phase, phase II. Under steady-state conditions an isotope effect of 2.0 was found on the overall electron transfer from L-lactate to ferricyanide. These transient reduction results were discussed in terms of a kinetic model implying intra- and inter-protomer electron exchanges between flavin and haem b2, all of which have been experimentally described. Computer simulations indicate that the reaction scheme provides a reasonable explanation of the fast-reduction phase, phase I (in absence of acceptor). The pseudo-first-order rate constant for oxidation of reduced haem b2 in flavocytochrome b2 increased with increasing ferricyanide concentration in a hyperbolic fashion. The limiting value at infinite ferricyanide concentration, which was attributed to the intramolecular electron-transfer rate from ferroflavocytochrome b2 to the iron of ferricyanide within a complex, was 920 +/- 50 s-1 at pH 7.0 and 5 degrees C. Stopped-flow and rapid-freezing measurements showed haem b2 and flavin to be 90 and 44% oxidized respectively under steady-state conditions in presence of ferricyanide. Simulation studies were carried out to check the participation of the proposed reduction sequence in the overall catalytic reaction together with the role of reduced haem b2 (Hr) and flavin semiquinone (Fsq) as electron donors to ferricyanide. When the rate of the intramolecular electron-transfer exchange between Fsq and ferricyanide was adjusted to 200 s-1, simulated data accounted for molar activities defined under various conditions of L-lactate, [2H]lactate and ferricyanide concentrations. Simulation studies were extended to data obtained using cytochrome c as acceptor and reaction catalysed by Saccharomyces cerevisiae flavocytochrome b2. The differences in reactivity observed for Hr and Fsq with ferricyanide and cytochrome c were discussed in terms of redox potentials, electrostatic interactions, distances and accessibility of the participating groups.

Canine cardiac sarcolemmal vesicles demonstrate rapid initial Na(+)-Ca2+ exchange activity.

To identify a rapid, uninhibited rate of exchange activity, we investigated in canine sarcolemmal vesicles the rapid kinetics of Na(+)-Ca2+ exchange. Sarcolemmal vesicles were incubated in 160 mM NaCl and 20 mM HEPES at 25 degrees C (pH 7.4) and actively loaded with 45Ca2+ for 2 minutes by Na(+)-Ca2+ exchange. After further uptake was inhibited by dilution into 0.15 mM Na(+)-free EGTA, sarcolemmal vesicles were immobilized on a rapid filtration apparatus that allowed millisecond resolution of 45Ca2+ fluxes. In the presence of external NaCl (Na+o) but not other monovalent cations (i.e., K+, Li+), a biphasic pattern of Ca2+ release was observed--an initial brief and rapid rate of Ca2+ release followed by a second slower, prolonged phase of Ca2+ release. Semilogarithmic plots of sarcolemmal Ca2+ content versus time were not linear but were consistent with a biexponential rate of Na+o-induced Ca2+ release during the first several seconds of the exchange reaction. The fast phase of Na+o-stimulated Ca2+ release was several thousand-fold more rapid than that in the absence of Na+o. Both phases of Ca2+ release showed a similar Na+o dependence (Km, approximately 12 mM) with evidence of a positive cooperative effect of Na+. Vmax of the fast and slow phases were approximately 37.0 and approximately 0.76 nmol/mg/sec, respectively. Using rapid-reaction techniques, we demonstrated in the present study that the initial velocity of sarcolemmal Na(+)-Ca2+ exchange activity is greater than previously reported in sarcolemmal vesicles and that this exchange process exhibits complex rate behavior with a biphasic pre-steady state kinetic pattern.

A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath)

1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol.

Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2.

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 degree C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 x 10(7) M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+.FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 = 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+. which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-pi-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.

Chemical kinetic measurements of a mammalian acetylcholine receptor by a fast-reaction technique.

In the presence of acetylcholine, the nicotinic acetylcholine receptor undergoes two rapid conformational changes: one in the 1-ms time region, leading to the formation of a transmembrane channel and signal transmission between cells, and the other in the 100-ms time region, leading to an inactive "desensitized" form with altered ligand-binding properties. To determine the properties of the receptor that are relevant for channel opening and signal transmission, we have developed a cell-flow technique that allows measurements to be made with cells prior to receptor desensitization. Here we illustrate the usefulness of the technique. A wide concentration range of both a ligand that controls the opening of receptor channels (carbamoylcholine) and a receptor inhibitor (procaine) was used to measure the dissociation constant of the receptor site controlling channel opening (2.4 X 10(-4) M), the channel-opening equilibrium constant (5.5), the inhibition constant for procaine (5.8 X 10(-5) M), and the rate coefficients for two desensitization processes of 5 s-1 and 0.2 s-1. The cell-flow technique illustrated here is of interest because, by rapid-reaction techniques, it extends the chemical kinetic approach from investigations of reactions in solutions to investigations of many different receptors that exist in membranes of central nervous system cells and whose properties are not well known.

7] Methods for the study of superoxide chemistry in nonaqueous solutions

Current knowledge of the chemical properties of superoxide anion, O2-, is based on studies of superoxide reactions in aqueous and nonaqueous solution. The superoxide dismutases were postulated to play a protective role within the cell by reducing the steady-state levels of superoxide, a species that is inevitably produced in some unknown amount as a by-product of aerobic metabolism. In aqueous solution, studies of superoxide chemistry require specialized rapid reaction techniques because of the rapid, spontaneous disproportionation of superoxide in protic media. Interest in the reactivity of superoxide in nonaqueous media began with discoveries that electrochemical reduction of dioxygen in dimethyl sulfoxide yielded stable solutions of superoxide, and that crown ethers would solubilize KO2 in aprotic solvents and could be used to prepare stable solutions of superoxide for various uses. This chapter reviews the methods for the preparation of solutions of superoxide in nonaqueous, aprotic solvents and the uses of these methods for the study of superoxide chemistry in such media.

Electron transfer and ligand binding in metallo proteins

The multiple biological roles of metalloproteins may be illustrated by those macromolecules which are involved in the transport of dioxygen and its utilization for energy production. Reversible binding is the basic feature of oxygen carriers, such as hemoglobins or hemocyanins, and the stability of the complex as well as the dynamics of ligand binding to the metal(s) is modulated by the interactions with the proteins. Likewise, electron transfer between metalloproteins and/or the final electron acceptor, dioxygen, involves metal ions (Fe or Cu in most cases), but the specificity and rates of the processes in question are under protein control. The basic theme of this paper will be an illustration of the mechanisms of control exerted by the protein moiety on the intrinsic reactivity of the metal ions. It will be emphasized that the use of rapid reaction techniques is a very valuable approach to test the mechanism and to identify the intermediates involved in the control. For the sake of clarity, the regulatory action of the protein may be dealt with at a different level. At the tertiary level, the immediate environment of the metal ion controls the intrinsic rates of either ligand binding and/or electron transfer, as illustrated by kinetic experiments on myoglobin and azurin (taken as prototypes). In multisubunit proteins, the quaternary state of the macromolecule is involved in the control of reactivity. The classical allosteric model has been widely used to described cooperative effects in hemoglobin and hemocyanin; the general significance of the multiple quaternary state of a macromolecule will be illustrated by its applicability to cytochrome c oxidase, presenting a two-state kinetic model which accounts for some kinetic properties of this crucial enzyme.

The mechanism of ATP hydrolysis catalyzed by myosin and actomyosin, using rapid reaction techniques to study oxygen exchange.

During ATP hydrolysis, myosin or subfragment 1 catalyzes the exchange of oxygens between water and phosphate, so that on average, each product Pi molecule contains more than one oxygen atom derived from water. Using quenched-flow techniques, the exchange process in both ATP and Pi was studied in the rabbit skeletal muscle subfragment 1 ATPase. The exchange in protein-bound ATP (M*.ATP) and protein-bound ADP.Pi (M**.ADP.Pi) was followed as a function of time. The pattern of exchange follows closely a model in which M*.ATP + H2O and M**.ADP.Pi are interconverted directly, without any characterizable intermediate between these species. All oxygen atoms of Pi are equivalent with respect to loss to solvent, and elimination of water to re-form ATP occurs with a rate constant of 15 s-1 (at pH 8.0, I 0.015 M, 20 degrees C). This is the first direct measurement of the rate constant for the transformation of M**.ADP.Pi to M*.ATP. Oxygen exchange during steady state ATP hydrolysis in the presence of acto-subfragment 1 also fits well to this model, with actin reducing the time available for M*.ATP and M**.ADP.Pi to undergo exchange.