METHODS OF INVESTIGATION OF THE STRUCTURE AND ACTION MECHANISMS OF ACTIVE SITES OFENZYMES - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 32

for the substrate. This idea was supported by the finding of effective inhibitors called
“transition state analogs” Transition state theory for enzymatic reactions proposes that
the rate enhancement imposed by enzymes is due to the tight binding or stabilization of
the activated complex relative to initial reagents. Knowledge of the TS can provide
information to design stable TS analogs. Such an approach has three important aspects:
(1) hinting to chemists about the plausible structure of specific inhibitors for synthesis,
(2) using these inhibitors to test working hypothesizes about TS structures, and (3) using
these inhibitors for the regulation of enzymatic processes in vitro and in vivo.
A computational method of the structure prediction of an inhibitor is based on an
analysis of the quantitative structure-activity relationship (QSAR) (Ariens, 1989: Martin
et al, 1996). In this method, quantities such as volume, hydrophobicity or a number of
specific groups are experimentally derived. QSAR for a given TS is a polynomial
equation with n terms. Each of these terms corresponds to the number of aforementioned
regions of a particular molecule under investigation. In the framework of this approach,
it is necessary to define, prior to synthesis and testing, a functional relationship between
molecular structure and molecular action. Then the polynomial equation can be used to
predict the inhibition constant of molecules that have been not tested experimentally.
Braunheim and Schwartz (1999) used ab initio quantum mechanics to investigate
molecules in transition states. Molecules in enzyme active sites are described as
coincidently oriented van der Waals surfaces that vary in geometry and electrostatic
potential. The theory takes into consideration that the energy of ionic interactions and
hydrogen bonds drops off with 1/r and van der Waals interactions drop of with As
a result, the relative geometric position of groups is important for the task of simulating
molecular recognition. The authors stressed that analysis of the quantum mechanical
wave function in the system is important for this recognition because the molecular
interactions are sensitive to subtle variations caused by intra- and intermolecular
polarization. Polarization across conjugated bonds and of large atoms such as Br and I
can have profound effects on binding.
The quantum description of molecules was created in the following way (Braunheim
and Schwartz, 1999): 1) the molecular structures were energy minimized using
semiempirical methods; (2) the wave function for the molecule was calculated; (3) from
this wave function, the electrostatic potential was calculated at all points around and
within the molecule: (4) the electron density, the square of the wave function, was
calculated; (5) with this information the electrostatic potential (EP) at the van der Waals
surface was generated. Regions with EP close to zero, a partial EP positive or negative
EP, and even greater potentials, may be involved in the van der Waals, hydrogen bonds
or in coulombic interactions, respectively.
The theoretically predicted values of ligand-binding free energy for
cytodine deaminase agreed with the values from experimental Thus, for the
citidine transition state and for a strong inhibitor
hydrated pyrimidine-2-one ribonucleoside and and for a
weak inhibitor uridine and Fig. 1.13 shows the
structures of the AMP nucleosidase transition state and of inhibitor structures, which
were theoretically predicted. The equilibrium constant of AMP was estimated as
The strongest inhibitor, formycin has the inhibitor constant