Initial velocity patterns intersecting

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

Equation 6 describes an intersecting initial velocity pattern where 1/v is plotted versus 1/A at different values of B, whereas Equation 6 describes the pattern where 1/v is plotted versus 1/B at different values of A (Fig. 1). Both the slopes and the intercepts of the reciprocal plots are functions of the other substrate concentration, and replots of slopes or intercepts versus the reciprocal of the other substrate concentration allow determination of all kinetic constants. 

Figure 1 Reciprocal plots with A varied at different levels of B (intersecting initial velocity pattern) or different levels of I (noncompetitive inhibition pattern).

Thus, the initial velocity pattern will be intersecting regardless of whether A and B, A and C, or B and C are the variable and changing fixed substrates. In each case, the third substrate would be held at constant concentration for the entire pattern. If substrate B is truly saturating, however, the reversible sequence is broken and the A-C initial velocity pattern becomes a parallel one. In practice, the slope effect becomes smaller and smaller as fi is raised, but unless B is raised to over 100 times the Michaelis constant, a parallel pattern will not be seen. The A-B and B-C initial velocity patterns will always be intersecting, regardless of the level of the other substrate. Tfie same pattern seen for the ordered mechanism is seen for one where A and B have to be added in that order, but C can be added randomly. Such mechanisms are known (Viola Cleland, 1982). 

While an ordered trisubstrate mechanism shows a parallel initial velocity pattern when substrate B is saturating, a completely random mechanism shows intersecting patterns at all times. If one substrate must be added first, but the other two can be added randomly (Section 123), parallel initial velocity pattern wiU be obtained when either B or C is saturating. This is easily understood if one remembers that the saturation with B leads to addition in order A, B, and C, while saturation with C causes the order to be A, C, and B, that is, saturation at the branch point diverts aU reaction flux through one path or the other. 

Note that the case (5) is a subcase of (4). Cases (4)-(6) cannot be distinguished easily, because in each case the initial velocity patterns will be intersecting and look very much like the ordered mechanism. Only the equilibrium ordered mechanism will give different initial velocity patterns. 

Based on isotope effects only, it is not possible to distinguish the Rapid Equilibrium Ordered from the Rapid Equilibrium Random mechanism. However, the first mechanism gives a distinctive initial velocity pattern that intersects on the ordinate with B as the varied substrate. To teU the difference between the Rapid Equilibrium Random and the Steady-State Random mechanism will require other methods, such as the isotope trapping method (Rose et al, 1974), or isotopic exchange. 

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. 

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 ... 

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