Kinesin Takes One 8nm Step per ATP Molecule Hydrolyzed

Kinesin Motility Assay

The molecular motor kinesin is an energase that transduces the Gibbs free energy of ATP hydrolysis into step-wise translocations along microtubules (MTs). Head-to-tail assay for kinesin motility. As a point of reference, a single kinesin molecule pushing a microtubule (radius 15 nm length < 20 mm) at a 1-m-m s rate exerts a force of 5 pN (Hunt et al., 1994). assay for kinesin motility. As a point of reference, a single kinesin molecule pushing a microtubule (radius 15 nm length < 20 mm) at...

Initial Rate Kinetics of Multi Substrate Enzyme Catalyzed Reactions

In enzyme kinetics, the terms two-substrate and three-substrate mechanisms refer to those, for which the reaction rate v depends on the concentrations of two and three substrates, respectively. Most enzymes actually catalyze reactions involving two or more substrates, and, with the obvious exception of isomerases, many one-substrate enzymes actually have two substrates. Examples are proteases and peptidases (Reaction Polypeptidem+n + H2O Fragmentm-COOH + NH2-Fragmentn, where the subscripts m...

Global Analysis Offers Added Advantages in Statistical Analysis

Although statistical analysis is vital for all kinetic data, traditional approaches focused on regression analysis of a data set are comprised of measurements from a single run. This approach was necessary largely because early digital computers had limited-capacity central processing units, and only one or at most two least-squares fitting routines could operate simultaneously. Today's PCs, however, are powerful enough to conduct simultaneous least-squares fitting of multiple data sets to...

Info

FIGURE 4.4 Plot of single-point assay of y-glutamylhydroxamate formation versus L-glutamine concentration for the transferase reaction catalyzed by Escherichia coli glutamine synthetase. ADP, hydroxy-lamine, arsenate, magnesium chloride, HEPES buffer (pH 7.8), and KCl were present at 1.5 mM, 100 mM, 30 mM, 30 mM, 40 mM, and 100 mM, respectively (temperature 37 C). Glutamine was varied from 2.5 mM to 25 mM. The final concentration of enzyme (state of adenylylation 1.5) was 4 mg mL, and the...

Abortive Complex Formation Alters Idealized Product Inhibition Patterns for Two Substrate Kinetic Mechanisms

In what was the first product inhibition study to test the Alberty treatment, Fromm and Nelson (1961) examined the kinetics of the Klebsiella pneumoniae ribitol dehydrogenase FIGURE 8.25 Predicted product inhibition patterns for the Ping Pong Bi Bi kinetic mechanism in the absence of abortive complex formation. NOTE The four lines through the data points in the upper two plots are obtained by holding the concentrations of product inhibitor P at zero (bottom line), X, 2X, and 3X, where X is the...

Nucleophilic Substitution is a Widely Used Reaction Mechanism in Enzyme Catalysis

Nucleophilic substitution reactions are intrinsically heterolytic in that the entering nucleophilic reagent carries a free pair of electrons to the reaction center. This electron pair forms a new bond, and the leaving group (the exiphile or nucleofuge) exits with an electron pair. Removal of an unstable leaving group is so thermodynamically disfavored that nucleophilic reactivity is almost invariably suppressed (March, 1992). The weakest bases are the best leaving groups, because they are the...

Negative Cooperativity Distinguishes KNF Models from MWC Models

The term negative cooperativity describes the condition wherein binding of a ligand molecule decreases a protein's affinity for subsequent molecules of ligand (Levitsky and Koshland, 1969). Negative cooperativity cannot occur in the Monod-Wyman-Changeux allosteric transition model, because a single dissociation constant defines ligation at all equivalent sites, both for the exclusive and non-exclusive cases. Therefore, any experimental observation of negative cooperativity in ligand binding...

Enzyme Electrodes Combine the Specificity of Biological Catalysis with the Versatility of Potentiometry or Amperometry

Analytical biochemists have successfully coupled ampero-metric and potentiometric devices to enzyme-catalyzed reactions to detect and or quantify an extraordinary range of substances (Bott, 2004 Cardosi, 1990 Lowe, 1989 Ryan, Smyth and Fagain, 1994). The output is a current, which is then detected, amplified, and reported in units of analyte concentration. Enzyme electrodes have proved to be remarkably versatile devices, especially when combined with microprocessor-based circuitry for...

Dark Field Microscopy Affords Direct Observation of Microtubule Assembly Disassembly Dynamics

Hotani and Horio (1986) used dark-field microscopy to observe stochastic microtubule (MT) growth and shortening, but technically speaking, the scattering behavior giving rise to the dark-field images required the gain loss of FIGURE 12.9 Illumination of microscopic objects by dark-field microscopy. Yellow lines are ray tracings. Most of the incident light rays are blocked by central region (black), the annular stop. Arrangement of lens permits only oblique, side-on illumination of the specimen....

N

FIGURE 8.49 Flow-chart describing post-screening characterization of lead compounds. Based on Copeland (2005). Note the structural similarity of the drug's nitrogen-containing heterocycle and the guanine ring within cGMP, as well as the sulfonamide and cyclic phosphodiester linkages. Early lead molecules exploited these structural features. Therefore, rather than applying Copeland's criteria to the original lead compound, the detailed action of sil-denafil will be considered here. Criterion-1....

Design Of Initialvelocity Enzyme Assays

The ideal initial-rate enzyme assay is specific, accurate, sensitive, convenient, and continuous. Despite the inherent stability of steady-state processes, the initial-rate phase -that period of a reaction over which there is a constant change in product concentration per unit time - can be surprisingly fleeting. A useful operational definition is that the initial-rate phase is the interval of time, over which the reaction rate v remains constant, with depletion of less than Copyright 2010, by...

The Concept Of A Reaction Mechanism

The chief ambition of enzyme chemists is to obtain the most complete description possible of an enzyme-catalyzed reaction. An enzyme's overall catalytic mechanism may be subdivided into four parts 1. Chemical Mechanism - A reaction scheme showing all bond-breaking -making steps, rearrangements, transition state(s), as well as the stereochemistry of partial and overall reactions. 2. Kinetic Mechanism - A scheme accounting for the time-dependent accumulation and breakdown of each enzyme-bound...

Cleland Scheme For Ordered Bi Bi Mechanism

In this scheme, called the Iso Uni Uni mechanism, product release does not immediately regenerate the free enzyme E. Instead, intermediate species F must isomerize to regenerate E. The intermediate species may represent an enzyme conformation or a protonated or deprotonated enzyme form that must lose or gain a proton. In other cases, an active-site proton may need to be relocated to restore an enzyme form capable of substrate binding. Note Use of the term Iso to describe a kinetic mechanism has...

Mechanismbased Inhibition

Although affinity labeling agents have been widely employed both as tools in enzyme chemistry and as therapeutic agents, they suffer the major disadvantage that their reactive groups frequently react with enzymes rather than the intended target. A better way for creating highly specific enzyme inhibitors is to rely on the catalytic mechanism of the target enzyme to generate a chemically reactive species If the resulting highly reactive electrophile is appropriately situated so as to undergo...

Ag

FIGURE 11.13 Effect of changes in the magnitude of ligand dissociation constants on the ligand saturation curves for a dimeric protein obeying the sequential transition model. In the KNF model, positive cooperativity is the consequence of successive incremental increases in binding energy (i.e., AG1 < AG2 < AG3 < DGn) negative cooperativity is the consequence of successive incremental decreases in binding energy (i.e., AGt > AG2 > AG3 > AGn), and independent binding, where AGt AG2...

Mechanoenzymes Catalysis Force Generation and Kinetics

While many organisms can live without oxygen, none can survive without generating force. And were we to search for life on some far-off planet totally devoid of carbon, we would surely find that, more than any other biotic signature, force generation is the common thread linking all self-perpetuating life forms. Mechanoenzymes power cellular locomotion, proliferation, and irritability - the telltale signs of life. ATP-dependent cytoskeletal motors generate locomotive forces. The...

Graphical And Quantitative Analysis Of Bisubstrate Kinetics

On the basis of the preceding sections, what becomes clear is that double reciprocal plots of initial-rate data cannot alone distinguish potential two-substrate mechanisms. Three bisubstrate kinetic mechanisms (e.g., Rapid Equilibrium Random Bi Bi, Steady-State Ordered Sequential Bi Bi, and Theorell-Chance Bi Bi) yield pairs of double-reciprocal plots that intersect to the left of the 1 v-axis. The rapid-equilibrium ordered mechanism stands alone among sequential mechanisms, with one double...

Determining The Rates Of Enzyme Synthesis And Degradation

Schoenheimer (1942) first recognized that enzyme and protein turnover is an essential process in which cellular constituents are continually replaced through protein biosynthesis and degradation. Turnover rids living cells of (a) proteins that are metabolically unnecessary, such as those remaining after a shift in nutrient availability or a diurnal change in metabolism (b) proteins that misfolded and are thus unable to function properly (c) proteins that racemized, such as those formed through...

B Reactions that are Unimolecular in Forward Direction and Bimolecular in Reverse Direction

We now consider the slightly more complicated case of a simple chemical reaction in which species A converts to B and C and vice versa Note further that the binomial term on the right-hand side of Eqn. 10.13 will give rise to (ax) however, because ax is taken to be small, the quantity (a x)2 will be very small for a system near equilibrium, then 0 kia kiDx kixe k_ i xe

Substrate Channeling

Observing that the intracellular concentrations of enzymes are in many cases surprisingly high, far above those used in most kinetic experiments, Srere (1985) suggested that functionally related enzymes are apt to interact and form noncovalent, multi-enzyme complexes that he termed metabolons. He further predicted that these supramolecular complexes may be endowed with special properties, particularly the ability to increase metabolic flux by mechanisms allowing scarce pathway intermediates to...

R

TABLE 2.18 Enzymes Catalyzing Reactions Involving Carbanion Intermediates Amine Dehydrogenase Catalyzes reaction of a primary amine (such as methylamine, n-propylamine, n-butylamine, benzylamine, (EC 1.4.99.3) and n-nonylamine) with water and an acceptor substrate to produce an aldehyde, ammonia, and the reduced acceptor. Tryptophan tryptophylquinone-containing amine dehydrogenases display Ping Pong Bi Bi kinetics, with the aldehyde released before the acceptor substrate binds. The first...

Ch2

S-(Aminoethyl)-Cysteine Mutant The resulting aminoethylated Lys-116-Cys enzyme had a kinetic pKa of 5.9. Such findings convincingly support the original proposal that the pKa of the Schiff base forming Lys-115 is decreased by repulsive electrostatic interactions with Lys-116. 7.3.5. pKa Values may be Estimated on the Basis of Protein Structural Calculations Most data on the ionization of functional groups within proteins and enzymes are experimentally obtained by the techniques listed in Table...

The Cahn IngoldPrelog System Allows One to Assign the Absolute Stereochemical Configuration of Chiral Compounds

This self-consistent set of rules (also known as the RS-system) specifies the absolute three-dimensional configuration ofchiral molecules (Cahn, Ingold and Prelog, 1966). The following abbreviated rules form the basis for the IUPAC systematic naming of compounds containing asymmetric atoms. Step-1. Define Substituent Priority. List substituents directly attached to the stereogenic carbon (or phosphorus within phosphates and phosphate esters) in decreasing order of atomic number Z. The atom of...

Egs

FIGURE 9.11 Free energy diagrams illustrating the kinetic regimes of undersaturation, saturation, and oversaturation. The changes for interconversion of free enzyme forms E1 and E2 are shown by the dotted lines. Reproduced with permission of the authors and the American Chemical Society. of synthesis and or interconversion of bound radiolabel to substrate and product, which upon release is unlikely to rebind. We can also evaluate the peak-switch concentration cp, corresponding to the degree of...

Chances Stopped Flow Technique Revolutionized the Investigation of Moderately Fast Reactions

To eliminate the need for large volumes of reactants and to increase the versatility of measurement, Chance (1943) invented the stopped-flow apparatus by adding a third stopping syringe to receive the mixed reactant solutions after passage through the mixer and observation cuvette (see Fig. 10.4). The two reactant syringes are attached to a pneumatic piston that is driven by compressed nitrogen gas. As the third syringe fills, its retreating plunger contacts an electronic switch, stopping the...

The Duke Le Novere and Bray Conformational Spread Model

The Conformational Spread (or CS) model describes cooperative behavior and ligand binding properties of oligomeric proteins assembled into closed-ring structures (Duke, Le Novere and Bray, 2001). Each functional subunit (or protomer) within a ring can only exist in either of two conformational states, designated as ( ) for active and (-) for inactive, that inter-convert rapidly and stochastically. Ligand affinity is taken to be higher for protomers in the ( ) conformational state, and the...

Coupled Or Auxiliary Enzyme Assays

When the enzyme reaction of primary interest produces no spectral change, it is convenient to devise a method whereby substrate depletion or product accumulation can be monitored spectrally by linking the reaction catalyzed by the primary enzyme to one or more auxiliary enzyme-catalyzed reactions. Such an arrangement allows a continuous enzyme Continuous Glucose-6-Phosphate Assay D-Glc + ATP D-Glc-6-P + ADP (No Spectral A) D-Glc-6-P + NADP+ 6P-Gluconolactone + NADPH TABLE 4.4 Troubleshooting...

Ly2121260

Gln-dependent Carbamoyl-P Synthetase YC-1 Potentiates NO activation of Soluble Guanylate Cyclase N-(5-fluorothiazol-2-yl)-3 (tetrahydropyran 4-yl)propionamide N-(5-fluorothiazol-2-yl)-3 (tetrahydropyran 4-yl)propionamide (Hill coefficient h z 1.7), the main mode of glucokinase control is thought to rest with its glucose insulin-induced biosynthesis, resulting in diurnal oscillation in GK concentration that matches in its high and low periods of feasting and fasting. In search of small molecules...

Several Key Properties of Allosteric Systems Suggested the Symmetry Conserving MWC Model

Mod Monod Wyman Changeux

Monod, Wyman and Changeux (1965) based their model on an extensive analysis available in the extant literature on allosteric systems, particularly the seminal observations of Umbarger and Brown (1957 1958) on threonine deaminase, Yates and Pardee (1956) on aspartate transcarbamoylase (ATCase), and Cohen et al. (1952) on the aspartokinases. In their original paper, they offered the following properties and definitions. Property -1. Homotropic effects are defined as cooperative binding...

Cleland Developed Useful Rules for Analyzing Reversible Dead End Inhibition

As noted earlier, whenever a dead-end inhibitor binds to an enzyme, catalysis cannot proceed. Because their action is well defined, dead-end inhibitors have many applications in the investigation of enzyme catalysis. Cleland (1963) offered the following rules for reversible, dead-end inhibition patterns, as observed in double-reciprocal plots of initial rate behavior. Rule-1. For a double-reciprocal plot of 1 v versus 1 A , if the vertical intercept varies with the concentration of the...

Aaa Mechanoenzymes

The AAA+ (standing for ATPases Associated with various cellular Activities) mechanoenzyme family is large and functionally diverse. These enzymes have the capacity to alter the conformation of specific target proteins and they play important roles in various cellular processes, including proteolysis, membrane fusion, cytoskeletal regulation, protein folding, and DNA replication (see Table 13.6). These energases are united by the presence of similar structural elements in their ATP binding...

C The Continuum between Sn1 and Sn2 Mechanisms

For solution-phase nucleophilic substitution reactions, the American physical organic chemist Sol Winstein treated Sn1 and Sn2 mechanisms as limiting cases that lie along a continuum of possible interactions (Scheme 9.21). N R-X N R+X- fe N R+IIX- N R+ + X- N R-X N R+X- fe N R+IIX- N R+ + X- N-R-X N-R+-X- N-R+IIX- N-R + X- In this scheme, R-X is the covalently bonded form, R+X is a so-called tight ion pair, R+ X is a loosely associated ion pair, and R+ + X represents the fully dissociated ions....

B

Symmetric Single-Minimum Energy Well FIGURE 2.5 Potential energy curves for proton transfer reaction along hydrogen bonds. A, Ordinary hydrogen-bonded complex, with an asymmetric single minimum (located at R0) for proton transfer B, Strong hydrogen-bonded complex, with a symmetric single minimum at R0 for proton transfer C, Strong hydrogen-bonded complex showing a symmetric double-minimum, again at R0, for proton transfer. The dotted curve indicates the lower potential energy barrier for proton...

Kinetics Of Enzymes Acting On Polymeric Substrates

Many enzymes act on polymeric substrates. Polymerases increase the degree of polymerization by catalyzing successive monomer addition reactions, whereas depolymerases reduce the degree of polymerization by catalyzing the breaking of bonds within polymeric substrates (Table 5.7). Polymerases generally act in an exo manner by catalyzing repetitive endwise addition reactions. On the other hand, depolymerases can act by exo and endo mechanisms, the latter resulting in internal scission to generate...

Product Inhibitors Often Provide Valuable Clues About Multisubstrate Iso Mechanisms

As noted in Chapter 3, while central complex isomerization has no effect on the form of the initial-rate equation, isomerization of stable enzyme forms (i.e., those where no chemical reaction occurs until another substrate is added) introduces additional terms into the rate equation. A great many enzymes form stable enzyme species that isom-erize. Peller and Alberty (1959) demonstrated that the magnitude of Vi Exotal can never exceed the magnitude of any unimolecular rate constant describing...

C Examples of Available Software

There are numerous published programs for simulating and analyzing enzyme rate data. Kineticists have always generously shared these programs, and acquiring them is now as simple as a few clicks of a computer mouse. For those interested in modeling the time-dependent changes of enzyme activity, such as the action of an irreversible enzyme inhibitor, the KINSIM and FITSIM programs have proven to be particularly useful. Frieden (1997) presented a general account of how this software allows an...

Role Of Atp In Protein Folding

The American enzyme chemist Christian Anfinsen was awarded the Nobel Prize in 1972 (see Table 1.2), for his demonstration in the early 1960s that the 124-residue polypeptide chain of pancreatic RNase folds spontaneously to form a native, compact enzyme possessing full catalytic activity. Even so, spontaneous folding of longer polypeptides cannot occur on biologically relevant time-scales (Gething and Sambrook, 1992). As pointed out by Ranson, White and Saibal (1998), cellular conditions of high...

The Actoclampin Hypothesis Concerning the Existence and Action of Cytoskeletal Filament End Tracking Motors

To solve the longstanding riddle of how actin polymerization might generate forces needed for cell crawling and other actin-based motile processes, Dickinson and Purich (2002) broke with the traditional assumption that free-ended filament elongation powers actin-based motility. They instead proposed the Actoclampin Molecular Motor Hypothesis asserting that all actin-based motility arises from forces generated by filament end-tracking motors. They argued that such tracking proteins mediate...

Bisubstrate Kinetic Mechanisms

As first suggested by Segal, Kachmar and Boyer (1952), valuable information on the kinetic mechanisms of Copyright 2010, by Elsevier Inc. All rights of reproduction in any form reserved. FIGURE 6.1 Preparation of reagents for the investigation of the substrate dependence of initial reaction rate v for a bisubstrate enzyme-catalyzed reaction. The diagram shows how 25 solutions may be prepared in a manner allowing one to determine the dependence over substrate concentration range that assures...

C Examples of Kinetic Solvent Isotope Effects

Hunkapiller, Forgac and Richards 1976 employed stopped-flow kinetics and proton inventory experiments have been used to define the reaction pathway for hydrolysis of a specific peptide substrate, Ac-L-Ala-L-Pro-L-Ala p-nitroanilide, by the serine proteases elastase and a-lytic protease. The stopped-flow studies reveal the existence and buildup of a tetrahedral adduct between the active-site serine hydroxyl group and the sensitive carbonyl group of the substrate. The decomposition of this...

Single Molecule Fluorescence Facilitates Observation of Dextran Binding to Bacterial Glucosyltransferase

Of the oral Streptococcal glucosyl transferases GTFs catalyzing glucosyl transfer from sucrose to growing glucan chains, GTF-I produces water-insoluble glucans by synthesizing a-1,3-glucans. As noted by Kaseda et al. 2000 , the presence of a-1,6-glucans greatly accelerates FIGURE 12.25 Single-molecule kinetics of dihydrofolate reductase. A, DHFR molecules viewed by total internal reflectance fluorescence microscopy. The length of each plume indicates the observed fluorescence intensity B,...