Initial velocity patterns equilibrium ordered

The third type of initial velocity pattern results from a mechanism in which 1) the substrates add in obligatory order and 2) the off-rate constant for the first substrate to bind exceeds the turnover number (V/Et or kcat) sufficiently that its binding is at equilibrium, which is called an equilibrium ordered initial velocity pattern (Fig. 3). The rate equation is... 

Figure 3 Reciprocal plots with B varied at different levels of A (equilibrium ordered initial velocity pattern) or different levels of I (competitive inhibition pattern).

This may change the initial velocity pattern, as in the case of fructose-6-sulfate, which is a slow substrate for phosphofructokinase. This substrate has lost sufficient affinity for the enzyme that it binds only when MgATP is present, and thus the mechanism changes from a random one with both substrates sticky with fructose 6-phosphate (fructose-6-P) to an equilibrium ordered one with MgATP adding first (19). Slow alternate substrates give cleaner and more easily interpreted pH profiles, and isotope effects are often (but not always) more fully expressed (see Sections VII,A and VII,B below). 

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 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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