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...

Info

Grubbs and Hoyeyda Ruthenium Catalysts Enantiomeric selectivity specificity FIGURE 1.2 Schematic of catalytic hydrogenation of ethylene on a nickel, platinum, or palladium surface. In this idealized representation, the metal surface acts as a rack, on which each of the reactants is stretched by binding to adjacent metal atoms. This physisorptive process occurs by interactions of reactant electrons with empty electron-deficient orbitals of the metal. Catalytic hydrogenation results from the...

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,...