Transition state enzyme mechanism Studies

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

In this chapter we have seen that enzymatic catalysis is initiated by the reversible interactions of a substrate molecule with the active site of the enzyme to form a non-covalent binary complex. The chemical transformation of the substrate to the product molecule occurs within the context of the enzyme active site subsequent to initial complex formation. We saw that the enormous rate enhancements for enzyme-catalyzed reactions are the result of specific mechanisms that enzymes use to achieve large reductions in the energy of activation associated with attainment of the reaction transition state structure. Stabilization of the reaction transition state in the context of the enzymatic reaction is the key contributor to both enzymatic rate enhancement and substrate specificity. We described several chemical strategies by which enzymes achieve this transition state stabilization. We also saw in this chapter that enzyme reactions are most commonly studied by following the kinetics of these reactions under steady state conditions. We defined three kinetic constants—kai KM, and kcJKM—that can be used to define the efficiency of enzymatic catalysis, and each reports on different portions of the enzymatic reaction pathway. Perturbations... 

Before our work [39], only one catalytic mechanism for zinc dependent HDACs has been proposed in the literature, which was originated from the crystallographic study of HDLP [47], a histone-deacetylase-like protein that is widely used as a model for class-I HDACs. In the enzyme active site, the catalytic metal zinc is penta-coordinated by two asp residues, one histidine residues as well as the inhibitor [47], Based on their crystal structures, Finnin et al. [47] postulated a catalytic mechanism for HDACs in which the first reaction step is analogous to the hydroxide mechanism for zinc proteases zinc-bound water is a nucleophile and Zn2+ is five-fold coordinated during the reaction process. However, recent experimental studies by Kapustin et al. suggested that the transition state of HDACs may not be analogous to zinc-proteases [48], which cast some doubts on this mechanism. 

In addition to these studies, the DHFR catalytic mechanism has been studied using the local density function (LDF) approach.22 A detailed understanding of the catalytic mechanism is often very useful in the design of high affinity enzyme inhibitors since it can shed light on the transition state structure and the key interactions used by the enzyme to stabilize it. 

The relationship between the geometry of the saddle point of index one (SPi-1) and the accessibility to the quantum transition states cannot be proved, but it can be postulated [43,172], To some extent, invariance of the geometry associated with the SPi-1 would entail an invariance of the quantum states responsible for the interconversion. Thus, if a chemical process follows the same mechanism in different solvents, the invariance of the geometry of the SPi-1 to solvent effects would ensure the mechanistic invariance. This idea has been proposed by us based on computational evidence during the study of some enzyme catalyzed reactions [94, 96, 97, 100-102, 173, 174, 181-184],... 

The Boltzmann distribution helps us understand how intermediates can become trapped in energy wells between successive transition states in a multistep reaction mechanism. This behavior forms the basis for a special field of enzymology known as cryoenzymology. By appropriate choice of water-miscible solvents, enzymes can be studied at ultra-low temperatures where the rates of interconversion of enzyme species can be greatly retarded. See Cryoenzymology... 

The substrate specificities of both mammalian and yeast hexo-kinases have been extensively studied (76,77). Nevertheless, work in this area continues both in the search for isoenzyme specific inhibitors and in increasingly detailed investigations of the catalytic mechanism. Recently potential transition state analogs PI-(adenosine-5 )-P3-glucose-6 triphosphate (Ap -glucose) and P1-(adenosine-5 )-P4-glucose-6 triphosphate (Ap.-giucose) were tested as inhibitors of four hexokinase isoenzymes. However, they were found to exhibit less affinity for the enzyme than either of the natural substrates alone (78). 

---