The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique.

Traditionally, enzyme transient kinetics have been studied by the stopped-flow and rapid quench-flow (QF) methods. Whereas stopped-flow is the more convenient, it suffers from two weaknesses: optically silent systems cannot be studied, and when there is a signal it cannot always be assigned to a particular step in the reaction pathway. QF is a chemical sampling method; reaction mixtures are aged for a few milliseconds or longer, 'stopped' by a quenching agent and the product or the intermediate is measured by a specific analytical method. Here we show that by exploiting the array of current analytical methods and different quenching agents, the QF method is a key technique for identifying, and for characterising kinetically, intermediates in enzyme reaction pathways and for determining the order by which bonds are formed or cleaved by enzymes acting on polymer substrates such as DNA.

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.

Kinetic method for differentiating mechanisms for ligand exchange reactions: application to test for substrate channeling in glycolysis.

We have derived analytical expressions for the kinetics of the two mechanisms involved in ligand substitution reactions. These mechanisms are (i) a dissociative mechanism in which the leaving ligand is first dissociated prior to the binding of the incoming ligand and (ii) an associative mechanism where a ternary complex is formed between the incoming ligand and the complex containing the leaving ligand. The equations obtained provide the theoretical basis for differentiating these two mechanisms on the basis of their kinetic patterns of the displacement reactions. Analysis of these equations shows that an associative mechanism can only generate an increasing kinetic pattern for the observed pseudo-first-ordered rate constants as a function of increasing concentration of the incoming ligand and plateaus, in most cases, at a value higher than the off-rate constant of the leaving ligand. However, a dissociative mechanism can generate either an increasing or a decreasing (kapp decreases with increasing concentrations of the incoming ligand) kinetic pattern, depending on the magnitudes of the individual rate constants involved, and, in either case, it will plateau at kapp equal to the koff of the leaving ligand. Therefore, the decreasing kinetic pattern is a hallmark for a dissociative mechanism. This general method was used to settle the dispute of whether NADH is transferred directly via the enzyme-enzyme complex between glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27).(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate channeling in glycolysis: a phantom phenomenon.

It has been proposed that glycolytic enzymes form multienzyme complexes for direct transfer of metabolites from the producing enzyme to the utilizing one. Reexamination of the supporting evidence, which involves the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, GPDH; EC 1.1.1.8) and with L-lactate dehydrogenase (LDH; EC 1.1.1.27), has shown that the supporting evidence is based on misinterpretation of the kinetics of ligand exchange. Srivastava et al. have responded with a revision of their own and criticism of our data. To clarify this problem, we have carried out detailed kinetic studies on NADH binding to GPHD and LDH and on the displacement of enzyme-bound NADH by LDH or GPDH. The experiments were conducted at 10 degrees C in 50 mM Hepes, pH 7.5/100 mM KCl/1 mM EDTA/1 mM 2-mercaptoethanol, using rabbit muscle GPDH and LDH. The results show that the kinetic patterns exhibited by the displacement of NADH-bound enzyme by either GPDH or LDH are consistent with a dissociative mechanism but not with a direct transfer mechanism. Theoretical analysis shows that a combined dissociative and direct transfer mechanism can explain the transient kinetic data reported by Srivastava et al. if, and only if, a majority (approximately 90%) of the enzyme present in lower concentration exists as a complex with the second enzyme. However, data from tracer and traditional sedimentation equilibrium and from gel filtration experiments show that LDH and GPDH do not form complexes in the presence of saturating NADH concentration when the enzyme concentrations are ranged between 4 and 50 microM, a concentration equal to or greater than that used by Srivastava et al. Our results demonstrate that GPDH and LDH do not form multienzyme complex and the transfer of NADH between these enzymes proceeds via a dissociative mechanism.

A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase.

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.

Reexamination of the kinetics of the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase and with lactate dehydrogenase.

Srivastava and Bernhard [Srivastava, D. K. & Bernhard, S. A. (1986) Science 234, 1081-1086] have proposed that glycolytic enzymes form multienzyme complexes for the direct transfer of metabolites from the producing enzyme to the utilizing one. We have reinvestigated the evidence for direct transfer of NADH between its complexes with alpha-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27). The results reveal the following. (i) Proper treatment of the kinetics of and equilibrium data for the transfer of NADH between GPDH and LDH indicates that NADH transfer proceeds by a free-diffusion mechanism and not by direct transfer through a ternary complex. (ii) The koff for NADH from its GPDH complex is 60 sec-1 rather than 9.4 sec-1 in Tris.HCl buffer (pH 7.4) at 25 degrees C. With this value one can explain kcat = 50 sec-1 for LDH-catalyzed hydrogenation of pyruvate with GPDH-bound NADH as coenzyme. (iii) Steady-state kinetics show that LDH inhibits the GPDH-catalyzed reaction simply by reducing the concentration of free NADH. Similarly, aldolase inhibits the GPDH-catalyzed reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by binding to the substrate. The proposed direct transfer of NADH between GPDH and LDH is therefore mainly based on a misinterpretation of the experimental data.

A transient kinetic study of enthalpy changes during the reaction of myosin subfragment 1 with ATP.

1. The enthalpy changes during individual reaction steps of the myosin subfragment 1 ATPase were studied with the use of a new stopped-flow calorimeter [Howarth, Millar & Gutfreund (1987) Biochem. J. 248, 677-682]. 2. At 5 degrees C and pH 7.0, the endothermic on-enzyme ATP-cleavage step was observed directly (delta H = +64 kJ.mol-1). 3. ADP binding is accompanied by a biphasic enthalpy change. 4. The release and uptake of protons was investigated by the use of two buffers with widely different heats of ionization. 5. Protons are involved in all four principal steps of the myosin subfragment 1 ATPase.

A stopped-flow calorimeter for biochemical applications.

A rapid-response stopped-flow calorimeter for small samples of reagents is described. The construction, performance characteristics and operational limitations are described, along with an example of its ability to resolve the kinetics of an enzyme-catalysed hydrolysis. It is thought likely that the method would find useful application in a variety of chemical and biochemical investigations.

Reflections on the kinetics of substrate binding.

The principle questions which are still being asked about enzyme-substrate complex formation are examined. Data for the maximum rates of collision complex formation are reviewed. The limitations of past experimental procedures and the potentialities of new approaches are discussed together with methods that allow the distinction between collision complexes and subsequent events.

Kinetics of acto-S1 interaction as a guide to a model for the crossbridge cycle.

Recent experiments on the kinetics of the interaction between myosin subfragment 1 (S1) and F-actin in solution are summarized. It is concluded that, at every step of the ATPase cycle, the association between the two proteins takes place in two stages. The equilibrium constant of the second step and thus the affinity of S1 for actin changes from step to step during the enzymatic reaction. It is proposed that the transient kinetic evidence can be interpreted in terms of two different classes of contraction models. The first one, which is widely used at present, identifies particular steps in the enzymatic reaction as directly responsible for the conformational change which represents the power stroke of muscle contraction (direct coupling model). In the second class of model, to which we wish to draw attention, changes in affinity modulated by the enzymatic reaction result in changes in the relative amounts of time spent by parts of the myosin molecule in two different environments. These environments determine whether the molecule exists in the 'long' or 'short' state, and it is the transition between these two which constitutes the power stroke (indirect coupling model).