Enzyme isotope exchange

Segel, 1. H., Emyme Kinetics Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience (1975). This book starts at the same elementary level as Biochemical Calculations and progresses to the modern subjects of steady-state kinetics of mullireac-tant enzymes, allosteric enzymes, isotope exchange, and membrane transport. 

This enzyme is of wide occurrence in bacteria where it is concerned with the reduction of nitrate and CO2 as well as sulphur. Methods for its estimation depend on measuring some activity of hydrogenase by (a) dye reduction (benzyl viologen or methylene blue), (b) isotopic exchange and (c) evolution of molecular hydrogen. Interpretation of quantitative results is difficult due to the complex relationship between the enzyme cell structure and the particular method selected. ... 

Figure 5.5 Activition of an [NiFe] hydrogenase such as that from D.gigas, by incubation with H2, as measured by different assay methods.These are production of H2 with the low-potential donor methyl viologen consumption of H2 with the high-potential acceptor dlchlorolndophenol and Isotope exchange of H2 with H2O. The proportions of the enzyme in three different states, unready, ready and active, are indicated by the shading.

Computer simulations also point to the regulatory potential of these non-productive complexes. See Deadend Complexes Inhibition Nonproductive Complexes Product Inhibition Substrate Inhibition Isotope Trapping Isotope Exchange at Equilibrium Enzyme Regulation... 

Selected entries from Methods in Enzymology [vol, page(s)] Equilibrium isotope exchange study of kinetic mechanism, 249, 466 site-directed mutagenesis of Escherichia coli enzyme, 249, 93 positional isotope exchange studies, 249, 423 product inhibition studies of three substrates three products reactions, 249, 207-208. 

Control of enzymic activity arising from the modulated access of substrates to a channel leading to the active site. Such a scheme was suggested for aspartate carbamo-yltransferase which has its complement of active sites located on the interior surface of the complex comprised of catalytic and regulatory subunits. Nonetheless, isotope exchange studies of this enzyme suggest that this form of enzyme regulation does not apply in the case of aspartate transcarbamoylase . ... 

A number of methods can assist in identifying and characterizing enol intermediates (as well as eneamine and carbanion intermediates) in enzyme-catalyzed reactions. These include (1) proton isotope exchange (2) oxidation of the intermediate (3) coupled elimination (4) spectrophotometric methods (5) use of transition-state inhibitors (6) use of suicide inhibitors (7) isolation of the enol and (8) destructive analysis. 

ENZYME RATE EQUATIONS (3. Derivation of Isotope Exchange Rate Equations )... 

A potential limitation encountered when one seeks to characterize the kinetic binding order of certain rapid equilibrium enzyme-catalyzed reactions containing specific abortive complexes. Frieden pointed out that initial rate kinetics alone were limited in the ability to distinguish a rapid equilibrium random Bi Bi mechanism from a rapid equilibrium ordered Bi Bi mechanism if the ordered mechanism could also form the EB and EP abortive complexes. Isotope exchange at equilibrium experiments would also be ineffective. However, such a dilemma would be a problem only for those rapid equilibrium enzymes having fccat values less than 30-50 sec h For those rapid equilibrium systems in which kcat is small, Frieden s dilemma necessitates the use of procedures other than standard initial rate kinetics. 

This enzyme [EC 3.1.3.9] catalyzes the hydrolysis of o-glucose 6-phosphate to yield o-glucose and orthophosphate. Some glucose phosphatases also catalyze transphosphorylation reactions from carbamoyl phosphate, hexose phosphates, pyrophosphate, phosphoenolpyru-vate and nucleoside di- and triphosphates, using D-glu-cose, D-mannose, 3-methyl-D-glucose, or 2-deoxy-D-glu-cose as phosphoryl acceptors. See Isotope Exchange (Reactions Away from Equilibrium)... 

Although most enzyme exchange studies have been investigated at equilibrium, the back exchange of labeled product while the reaction is proceeding in the forward direction can provide valuable information about enzymic catalysis. Under favorable conditions, one may utilize such isotope exchange data to learn about the order of product release and the presence of covalent enzyme-substrate compounds. One of the first systems to be characterized in this way was glucose-6-phos-phatase . ... 

Cleland introduced the net rate constant method to simplify the treatment of enzyme kinetic mechanisms that do not involve branched pathways. This method can be applied to obtain rate laws for isotope exchange, isotope partitioning, and positional isotope exchange. Since the net-rate constant method allows one to obtain VraaJKra and in terms of the individual rate constants, this method has greatest value for the characterization of isotope effects on and Kj. Because only... 

Selected entries from Methods in Enzymology [vol, page(s)] Pepsin Activation energy, 63, 243-245 isotope exchange, 64, 10 kinetic constants, 63, 244 kinin-releasing enzyme, 80, 174 porcine, homology to cathepsin D, 80, 578 spectrokinetic probe,... 

Positional isotope exchange ( PIX ) is a very valuable technique in determining enzyme mechanisms, particularly those utilizing ATP, GTP, or another NTP substrate -. For example, the nucleotide substrate can be labeled with in bridging (i.e., P—O—P or phos-phoanhydride oxygen) and/or its nonbridging positions. 

By using NMR in the presence of the enzyme, an investigator can identify bridged-to-nonbridge (and the reverse) isotope exchanges and thereby identify probable intermediates on the reaction pathway. The procedure is useful for any enzyme-catalyzed reaction in which the individual atoms of a functional group within a substrate, intermediate, or product become torsionally equivalent during the course of a reaction. 

An isotope effect technique applied in enzyme-catalyzed isotope exchange experiments measuring reaction rate as a function of the [D20]/[H20] ratio. Such studies often provide information concerning the effect of the environment of the transition state on exchangeable protons. 

ISOTOPE EXCHANGE AT EQUILIBRIUM WEDLER-BOYER TECHNIQUE CUMULATIVE INHIBITION UNCONSUMED SUBSTRATE CRYPTIC CATALYSIS BOROHYDRIDE REDUCTION ENZYME CASCADE KINETICS... 

Isotope exchange behavior of ping pong enzymes,... 

ORDERED Bl Bl ENZYME MECHANISM ISOTOPE EXCHANGE AT EQUILIBRIUM... 

Isotope exchange experiments with purified F reveal a remarkable fact about the enzyme s catalytic mechanism on the enzyme surface, the reaction ADP + P, ATP + H20 is readily reversible—the free-energy change for ATP synthesis is close to zero When ATP is hydrolyzed by Fi in the presence of 180-labeled water, the Pj released contains an 180 atom. Careful measurement of the 180 content of P, formed in vitro by Fx-catalyzed hydrolysis of ATP reveals that the P, has not one, but three or four 180 atoms (Fig. 19-21). This indicates that the terminal pyrophosphate bond in ATP is cleaved and re-formed repeatedly before P, leaves the enzyme surface. With P, free to tumble in its binding site, each hydrolysis inserts 180 randomly at one of the... 

Isotope exchange at equilibrium. Consider the reaction of substrates A and B to form P and Q (Eq. 9-51). If both reactants and both products are present with the enzyme and in the ratio found at equilibrium no net reaction will take place. However, the reactants and products will be continually interconverted under the action of the enzyme. Now if a small amount of... 

When 31P is bonded to lsO the chemical shift of the 31P is altered by 0.0206 ppm from that when the phosphorus is bonded to leO. This allows lsO labels introduced into phospho groups to serve as tracers which can be followed continuously during reactions.683 The technique is useful in studies of stereochemistry (see Section 2) and for examination of positional isotope exchange.690 This latter technique is often used with ATP containing lsO in the P,y-bridge position. If an enzyme transfers the terminal (y) phospho group to an acceptor via a phosphoenzyme but without loss of the ADP, we may expect positional isomerization. The lsO will move between the P,y-bridge position and... 

Chemical studies also support the indicated mechanism. For example, the P-oxoacid intermediate formed in step b of Eq. 13-48 or Fig. 13-12 has been identified as a product released from the enzyme by acid denaturation during steady-state turnover.273 274 Isotopic exchange with 3H in the solvent275 and measurement of 13C isotope effects277 have provided additional verification of the mechanism. The catalytic activity of the enzyme is determined by ionizable groups with pKa values of 7.1 and 8.3 in the ES complex.278... 

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