Initial velocity studies

Hyland, L., et al., 1991. Human immunodeficiency viru.s-1 protea.se 1 Initial velocity. studies and kinetic characterization of reacdon intermediates by Isotope exchange. Biochemistry 30 8441-8453. 

From Figure 4.54 it can he seen that the family of reciprocal plots obtained at different fixed concentrations of NADP are essentially parallel to one another. This is also indicated in Figure 4.55, where the value of the slopes of the tines seem to be approximately constant. These results imply that the velocity equation for the ping-pong mechanism [146] can be used to describe the rate of the reaction catalyzed by G6PDH. Although initial velocity studies alone cannot define the exact kinetic mechanism [146,147], we are more interested in the appropriate rate equation that describes the reaction progress. 

A number of initial velocity studies have been made with yeast hexokinase. All results to date indicate an intersecting initial velocity pattern in... 

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),... 

The equations for initial velocity studies of a rapid equilibrium ordered bireactant system are ... 

The compulsory ordered mechanism arrived at through isotopic exchange rates is basically in agreement with mechanisms postulated on the basis of initial velocity studies. Heyde and Ainsworth showed that for beef heart m-MDH the initial velocity pattern in the absence of products and the product inhibition pattern are consistent with an ordered mechanism (77). Raval and Wolfe obtained similar results with pig heart m-MDH data obtained by initial velocity studies in both directions are in agreement with a compulsory ordered mechanism (78,79). Substrate inhibition by oxaloacetate also occurs with pig heart m-MDH (80). Similar initial velocity studies on beef heart s-MDH did... 

It is quite clear that the initial velocity studies, in the absence of products, are not sufficient for the determination of the order of addition of reactants in ordered sequences, while product inhibition experiments with either of the products provides this information unambiguously. 

Therefore, initial velocity studies alone will not distinguish between the two mechanisms however, the missing ABP and BPQ denominator terms lead to different product inhibition patterns. 

Initial velocity studies can place all the mechanisms listed in Table 3 into only three groups (Ping Pong, sequential, and equilibrium ordered), while aU of these can be told apart by product inhibition analysis. This clearly illustrates the immense power of the product inhibition analysis. 

Equations (12.28) and (12.30) are symmetrical, but not Eq. (12.29). Thus, from the initial velocity studies one can identify the substrate B, but cannot tell apart the substrate A from the substrate C. Binding studies, however, will identify A as the only substrate which binds to the free enzyme. 

Initial Velocity Studies in the Absence of Added Products and Dead-End Inhibitors... 

Initial velocity studies where NAD was varied around its Kj at different fixed levels of DNA (Hae III digests of pBR322 or Hae III digests with terminal S P04 groups removed) around its give a pattern which intersects to the left of the vertical axis (Fig. la,b). This is most apparent in Fig. lb, where NAD concentrations of up to 40 times Kj were used. The data were fit to the theoretical equations for a sequential mechanism (v = VAB/Kj Ki, + K B + K A + AB) and for an equilibrium ordered mechanism (v = VAB/Kj H + K A + AB) using the FORTRAN programs developed by Cleland [4]. V represents and represent Kj values for A and B, Kj ... 

Initial velocity studies were conducted in the presence of a number of dead-end inhibitors of NAD and DNA binding. Nicotinamide, a product of the reaction which acts as a dead-end inhibitory analog of NAD, is competitive vs NAD (Fig. 2, cf. [3]) and noncompetitive vs DNA (not shown). NADH exhibits similar kinetic properties and is competitive vs NAD (not shown) and noncompetitive vs DNA (Fig. 3). Both monomeric and polymeric dead-end inhibitory analogs of DNA behaved similarly. Thymidine (Fig. 4) and poly(dT) (data not shown) are both competitive vs DNA. The polymeric form is seven times more effective as an inhibitor based on total thymine content (Kj values of 5 juM vs 35 juM)- On a per molecule of inhibitor basis the difference is even more striking, the poly(dT) being more than 1000 times more effective than thymidine. Both poly(dT) (Fig. 5) and thymidine (data not shown, cf [3]) also exhibited competitive kinetics vs NAD. 

Initial velocity studies of the reaction catalyzed by poly(ADP-ribose) polymerase have been carried out under a variety of experimental conditions. An initial velocity pattern where NAD is varied at different fixed concentrations of DNA intersects to the left of the vertical axis. Nicotinamide, a product of the reaction, is competitive vs NAD and noncompetitive vs DNA. Initial velocity studies using dead-end inhibitors show that NAD analogs are competitive vs NAD and noncompetitive vs DNA, while DNA analogs are competitive vs both DNA and NAD. These data are most consistent with a random mechanism (Fig. 6). For as yet unknown reasons, DNA analogs do not appear to bind to enzyme NAD. 

The patterns of product inhibition and initial velocity studies suggested that the reaction occurred by an ordered Bi Bi mechanism [according to the nomenclature of Cleland (1963)J, in which the first substrate to bind to the enzyme and the last product released show competitive inhibition between each other. In this particular case, UTP would be the first substrate to add to the enzyme, and UDP-glucose would be the last product released, as outlined below ... 

The kinetic mechanisms of PP-ribose-P synthetase partially purified from human erythrocytes has been studied. Double reciprocal plots of initial velocity studies with variable MgATP cencen-trations and fixed ribose-5-P are intersecting and suggest a sequential kinetic mechanism, (Figure 2). Product inhibition studies... 

Fig. 2. Double reciprocal plot of initial velocity studies with variable MgATP concentrations and fixed ribose-5-P concentrations ranging from 2 to 10 juM. Magnesium concentration is 6 mM. Inset shows a secondary plot of the slope ( - ) and the intercept ( — ) versus the inverse of ribose-5-P concentration in micromolar. The Km for MgATP is 14 pM and ribose-5-P is 33 liM (From Fox and Kelley, 1972).

Initial velocity studies with magnesium complicate this postulated mechanism by implicating a separate sequential binding of this divalent cation to PP-ribose-P synthetase, in addition to its well known complex formation with ATP. 

---