Enzyme-bound equilibrium mixtures

Fig. 9. > P-NMR spectra (at 40.3 MHz) of the argmine kinase reaction (Nageswara Rao et al 1976) at 12°C and pH 7.25. (A) Equilibrium mixture ([nucleotide]/[E] = 4000) of overall reaction, catalytic concentration of enzyme. (B) Equilibrium mixture ( nucleotide]/[E] =0.97) of enzyme-bound substrates and products. (C) Same as (B) with EDTA added to chelate Mg and stop reaction, no chemical exchange. Shifts upfield are negative.

It is reasonable to identify the intermediate indicated by the above-mentioned experiments as a y-glutamyl-enzyme compound, an interpretation not excluded by any of the experimental results. There is, however, another plausible explanation for the observations, which does not necessarily involve a covalent enzyme-substrate compound of this kind. In this alternative proposal the rate determining steps in the catalytic reaction are not involved with the covalent bond processes but are conformational changes in the enzyme-substrate and enzyme-product complexes. If product is not released from the enzyme until a large number of rapid covalent reactions with the available nucleophiles has occurred, then any substrate will be converted to the same equilibrium mixture of bound products (e.g., glutamic acid and glutamyl hydroxamic... 

The binary complex can exist as an equilibrium mixture of a number of species as shown in Fig. 43. The major species apparent in a number of binary complexes, e.g. glutamate.decarboxylase and aspartate aminotransferase [31,90] appear to be those absorbing at 420 and 333 nm which are attributed to structures 2 and 3 (Fig. 43) respectively as expected the ratio of these species is pH dependent. On the other hand, the ternary complex may exist as an equilibrium mixture composed of species from both binary and ternary complexes, thus producing a composite electronic absorption profile however, the ternary complex of aspartate aminotransferase exhibits only two major absorption maxima at 430 and 340 nm due to substrate-coenzyme Schiff base and enzyme bound pyridoxamine-P respectively [90]. It is interesting to note the spectra observed for aspartate aminotransferase... 

Experimental results obtained by using P NMR of enzyme complexes of some specific enzyme systems are described in this section. These results are presented in two categories (1) inert enzyme-bound complexes of substrates, inhibitors, cofactors, and their analogs, and (2) equilibrium mixtures in the enzyme-bound form (i.e., where the substrates and products arc observed as they interconvert on the enzyme). The studies include a number of different enzymes however, the list is not complete. The enzyme systems chosen for description are expected to illustrate the experimental and theoretical considerations presented in Section II and indicate the kind of information that might be extracted by P-NMR studies of enzyme complexes and the limitations and difficulties of the method. 

Fig. 10. P-NMR at (40.3 MHz) spectra of an equilibrium mixture (E + ATP + AMP + MgCy of substrates and products fully bound to porcine adenylate kinase (Nages-wara Rao et al, 1978a) at 15°C and pH 7.0. (A) After 4 h of accumulation. (B) As in (A), but after —12 h separate resonances for each ADP bound to enzyme. (C) As in (B), but with addition of exogenous ATP. (D) As in (C), but with EDTA added to stop reaction. Shifts upheld are negative.

A P spectrum of an equilibrium mixture of the activation reaction of this enzyme [Eq. (8)] is shown in Fig. 12. An ambiguity existed initially regarding the position of the enzyme-bound Met-AMP in this spectrum. The P resonance of chemically synthesized Met-AMP was found to be —7.5 ppm at pH 4.1. In order to arrive at the assignment shown in Fig. 12A,... 

Fig. 13. Comparison of experimental (at 40.3 MHz) and computer-calculated P-NMR spectra for the equilibrium mixture of enzyme-bound substrates and products of arginine kinase (12 C pH 7.25). (A) Experimental spectrum (Nageswara Rao et al., 1976). (B) Computer-simulated spectra with a phosphoryl transfer rate of 120 s >. Shifts upheld are negative. From Vasavada et al., (1980).

Binding of a reversible inhibitor to an enzyme is rapidly reversible and thus bound and unbound enzymes are in equilibrium. Binding of the inhibitor can be to the active site, or to a cofactor, or to some other site on the protein leading to allosteric inhibition of enzyme activity. The degree of inhibition caused by a reversible inhibitor is not time-dependent the final level of inhibition is reached almost instantaneously, on addition of inhibitor to an enzyme or enzyme-substrate mixture. 

Table 8.6, entry d). The effect of CAII on the equilibrium of the dynamic mixture was significandy reduced in the presence of 8.116, which bound the enzyme and reduced its template effect. 

It should also be considered that the formation of the complex between activator and lipid is an equilibrium reaction with a finite dissociation constant. Under the conditions used for the quantification of activators— that is, with pure glycolipid substrates at concentrations well above the Kq of the respective activator-lipid complex—the activator can be assumed to be saturated with the lipid, so that the activator concentration practically equals the concentration of the substrate of the reaction (the activator-lipid complex). However, the presence of other lipids such as phospholipids in the assay mixture may increase the experimental Kd by orders of magnitude since the mixed aggregates formed may be much more stable than the pure glycolipid micelles. (At a large excess of phospholipids as in the case of liposome-bound substrate, the may depend linearly on the phospholipid concentration.) As a consequence the concentration of the activator-lipid complex may be far below the total activator concentration, and the enzymic reaction will accordingly be much slower than with pure glycolipid substrates. 

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