Structural definition, molecular mechanics

In this chapter, it will be described how we have utilized endogenous and engineered Zn2+ binding sites to explore the structure and molecular function of Na+/Cl -depen-dent neurotransmitter transporters. The work has not only allowed the definition of the first structural constraints in the tertiary structure of this class of transporters, but also provided new insight into both conformational changes accompanying substrate translocation and mechanisms governing conformational isomerization in the translocation cycle. In the chapter, we will also review the theoretical and practical basis for... 

In the absence of definitive information about the structure of the active site theoretical modeling of enzyme catalyzed reactions is difficult but not impossible. These difficulties are caused by the extremely large size of the enzyme-substrate-solvent system which typically comprises thousands or tens of thousands of atoms so that direct theoretical treatment at the microscopic quantum mechanical level is not yet practical. The computational demand is simply too enormous. As a compromise, a scheme generally referred to as QM/MM (quantum mechanics/molecular mechanics) has been devised. In QM/MM calculations, the bulk of the enzyme-solvent system (i.e. most of the atoms) is treated at a low cost, usually at the molecular mechanics (MM) level, while the more nearly correct and much more expensive quantum level (QM) computation is applied only to the reaction center (active site). 

The solution structures of a number of metalloproteins with paramagnetic metal centers were determined with molecular mechanics and dynamics in combination with NMR spectroscopy (see also Chapter 9)1124-126,189]. Due to the complexity of the molecules, for metalloproteins a crystal structure of the compound or a derivative is often needed for the definition of the starting geometry. Molecular dynamics is then used to find low-eneigy conformers. The dynamics calculations also allow the visualization of areas of large flexibility, and this may lead to some understanding of the enzyme mechanism11891. 

The connection between molecular mechanics and crystal structures came about in the attempt to quantify the non-bonded interactions. These were first taken oyer from intermolecular interaction potentials of rare-gas-type molecules. They start from the premise, contained in the van der Waals equation of state for real gases, that atoms are not localized at points, i.e. not at their respective nuclei. They occupy a volume of space and can be assigned, at least as a first step, more or less definite radii, by custom called van der Waals radii, which were initially estimated for many types of atom mainly from packing radii in crystals. Mutual approach of non-bonded atoms to distances less than the sum of these radii leads to strong repulsive forces. The empirical atom-atom potentials that were introduced to describe the balance between atom-atom attractions and repulsions were assumed to be characteristic of the atom types and independent of the molecules they are embedded in. They were assumed to hold equally for interactions between non-bonded atoms in... 

Before getting more involved in the finer points of particle adhesion studies by molecular mechanics it must be pointed out that such an approach suffers from considerable drawbacks. Molecular mechanics and dynamics by definition involve interactions between clearly defined types of atoms, with clearly defined atomic characteristics, placed in clearly defined molecular structures. Thus, a generalized, fictitious surface of only, let s say nitrogens, or even worse of generalized spheres, is a rather extreme physical approximation. It is by definition incorrect in a molecular mechanics and dynamics investigation. Such a drawback needs to be pointed out to put in perspective and understand... 

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