Steady-State Ping Pong mechanism

One less kinetic parameter can be obtained from an analysis of the data for a ping-pong mechanism than can be obtained for ordered reactions. Nevertheless, in Eq. 9-47, twelve rate constants are indicated. At least this many steps must be considered to describe the behavior of the enzyme. Not all of these constants can be determined from a study of steady-state kinetics, but they may be obtained in other ways. 

LiP catalyzes the oxidation of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) to veratryl aldehyde. Since this reaction can be easily followed at 310 nm, it is the basis for the standard assay for this enzyme (26,27). The enzyme exhibits normal saturation kinetics for both veratryl alcohol and H202 (28,43). Steady-state kinetic results Indicate a ping-pong mechanism in which H202 first oxidizes the enzyme and the oxidized intermediate reacts with veratryl alcohol (43). The enzyme has an extremely low pH optimum ( 2.5) for a peroxidase (43,44) however, the rate of formation of compound I (kx, Fig. 2) exhibits no pH dependence from 3.0-7.0 (45,46). Addition of excess veratryl alcohol at pH 3.0 results in the rapid conversion of... 

Steady-state kinetic analysis shows that biotin-dependent reactions proceed by way of a two-site ping-pong mechanism the two-part reactions are catalyzed at distinct sites in the enzyme. These sites may be on the same or different polypeptide chains in different biotin-dependent enzymes. The e-amino linkage of lysine to the side chain of biotin in biocytin allows considerable movement of the coenzyme - the distance from C-2 of lysine to C-5 of biotin is IdA, thus allowing movement of biotin between the carboxylation and carboxyltransfer sites. 

The steady state rate equation for this ping pong mechanism is Equation 4. 

Hexokinase does not yield parallel reciprocal plots, so the Ping Pong mechanism can be discarded. However, initial velocity studies alone will noi discriminate between the rapid equilibrium random and steady-state ordered mechanisms. Both yield ihe same velocity equation and families of intersecting reciprocal plots. Other diagnostic procedures must be used (e.g., product inhibition, dead-end inhibition, equilibrium substrate binding, and isotope exchange studies). These procedures are described in detail in the author s Enzyme Kinetics behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley-Interscience (1975),... 

Steady-state analysis of DPD in addition to crystal strucmres ° has led to the proposal of a two-site ping-pong mechanism, in which NADPH binds and reduces FAD at one end of the protein. The reducing equivalents are passed to the FMN at the other end of the protein via the Fe-S clusters. The reduced FMN reacts with the pyrimidine forming dihydropyrimidine and oxidized FMN. Here, we will focus on the pyrimidine reaction. 

The enzyme contains FAD as a prosthetic group and the steady-state kinetics implicate a ping-pong mechanism. Since dihydrofolate is a potent competitive inhibitor of NADPH-methylenetetrahydrofolate oxidoreduction, it was thought that... 

The steady state kinetics of arsenite oxidoreductase from A. faecalis indicate a so-called double displacement (or ping-pong ) mechanism (15) in which the enzyme cycles between oxidized and reduced forms in its reaction with arsenite and azurin (or cytochrome c). This overall kinetic scheme is common in redox-active proteins. Arsenite must bind, the oxygen atom transfer chemistry take place, and arsenate dissociate before the subsequent reaction of a second molecule of substrate. Since arsenate is not an inhibitor of arsenite oxidoreductase (43), product dissociation must be effectively irreversible. The turnover number (kcai) of 27 sec and for arsenite of 8 pM are reasonable parameters for the detoxification of arsenite, especially since A. faecalis is able to survive in at least 80 mM (1%) sodium arsenite. The considerable catalytic power of the enzyme is reflected by the kinetic parameter k JK of 3.4 X 10 M sec , which is fairly close to the diffusion-controlled maximum of 10 -10 M sec for proteins in... 

The isotope effect on V/Kb, (Y/Kb), is equal to 1 due to lack of an isotope-sensitive step in the second half-reaction. If it is not known whether the kinetic mechanism is sequential or Ping Pong, the isotope effects on the V/Ka and V/Ks values will not distinguish between a Steady-State Ordered and Ping Pong mechanisms. 

In a sequential mechanism, an isotope effect equal to or close to one on one of the two substrate V/K values suggests a Steady-State Ordered mechanism. The V/K for the first substrate bound will have the isotope effect of unity. The isotope effect of one may also apply to the second substrate in a Ping Pong mechanism, but a distinctive, initial velocity pattern with parallel lines is obtained in this case. 

For this mechanism, the enzyme must bind substrate A first, followed by the release of product P and the formation of the enzyme species E. This is followed by binding of substrate B to E and the breakdown of the E B complex to free enzyme E and the second product Q. Thus, for ping pong mechanisms, no ternary complex is formed. A general steady-state scheme for this type of reactions is... 

A procedure used to assist in identifying sequential mechanisms when the double-reciprocal plots exhibit parallel lines ". In some cases, bireactant mechanism can have various collections of rate constants that result in so-called parallel line kinetics, even though the mechanism is not ping pong. However, if the concentrations of A and B are kept in constant ratio with respect to each other, a sequential mechanism in a 1/v v. 1/[A] plot would be nonlinear (since in the denominator the last term of the double-reciprocal form of the rate expression contains [A] for example, for the steady-state ordered Bi Bi reaction scheme in which [B] = a[A], the double-reciprocal rate expression becomes 1/v =... 

A mathematical equation indicating how the equilibrium constant of an enzyme-catalyzed reaction (or half-reaction in the case of so-called ping pong reaction mechanisms) is related to the various kinetic parameters for the reaction mechanism. In the Briggs-Haldane steady-state treatment of a Uni Uni reaction mechanism, the Haldane relation can be written as follows ... 

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