Enzyme, cleft mechanism

One result from the analysis of the MD simulation was the proposal of a new enzymic pathway for hydrolysis by lysozyme. We begin with a description of the alternative mechanism, and the basis on which it was proposed. The energetics of the individual GlcNAc units in the lysozyme cleft are then presented, followed by a graphical representation of the correlation between the atomic fluctuations of the substrate and those of the enzyme. Of particular interest is the fact that the binding interactions stabilize a bound state conformation for the two glycosides involved in hydrolysis that is optimum for catalysis by the alternative mechanism and which differs from the conformations of the other glycosides. These conformational features are described in the final two sections. 

Acetylcholine is a relatively small molecule that is responsible for nerve-impulse transmission in animals. As soon as it has interacted with its receptor and triggered the nerve response, it must be degraded and released before any further interaction at the receptor is possible. Degradation is achieved by hydrolysis to acetate and choline by the action of the enzyme acetylcholinesterase, which is located in the synaptic cleft. Acetylcholinesterase is a serine esterase that has a mechanism similar to that of chymotrypsin (see Box 13.5). 

The conformation of membrane-bound enzymes is undoubtedly restricted by the membrane. However, the mechanism of action of these enzymes appears to be similar to that of soluble enzymes, so that the presence of clefts and conformational flexibility is to be expected. The mitochondrial coupling factor apparently contains both the ATP synthesizing enzyme and a proton channel conformational changes undoubtedly play a role in the function of this system. A large movement of polypeptide chains has been proposed in the functioning of this system (and for other membrane-bound enzymes), but no convincing experimental evidence is available to support such a hypothesis. 

The mechanism can only be retained if a base very much better than water is present at the enzyme active site to deprotonate the Zn(OH2)2+ species. In fact, the structure determination reveals a histidine residue with its side chain positioned approximately halfway down the -15 A-deep cleft in the protein structure within which the Zn site is located. This arrangement could act as a proton shuttle between the Zn(OH2)2+ and external solvent water, possibly via another two water molecules also found within the cleft. As a consequence, the enhancement of ligand acidity by Zn11 is more important in the kinetic than the equilibrium sense (taken from http //www.chem.uwa.edu.aU/enrolled students/BIC sect4/sect4.2.htmll. 

FIGURE 19-1 T Mechanism of action of cholinergic stimulants. Direct-acting stimulants bind directly to the postsynaptic cholinergic receptor. Indirect-acting stimulants inhibit the cholinesterase enzyme, thus allowing acetylcholine to remain in the synaptic cleft. 

Acetylcholine is a neurotransmitter that functions in conveying nerve impulses across synaptic clefts within the central and autonomic nervous systems and at junctures of nerves and muscles. Following transmission of an impulse across the synapse by the release of acetylcholine, acetylcholinesterase is released into the synaptic cleft. This enzyme hydrolyzes acetylcholine to choline and acetate and transmission of the nerve impulse is terminated. The inhibition of acetylcholineasterase results in prolonged, uncoordinated nerve or muscle stimulation. Organophosphorus and carbamate pesticides (Chapter 5) along with some nerve gases (i.e., sarin) elicit toxicity via this mechanism. 

In short, the reaction mechanism consists of a dehydration, a flip, and a hydration. The first and the last steps appear to be well defined on the basis of spectroscopy, crystallography, and chemical common sense. The details of the flip, perhaps the key feature of the mechanism, are less clear. There are no crystals of the cis-aconitate complex (in fact, there should be two different complexes, 5 and 7). The free-enzyme intermediate 6 has not been isolated. Displacement of one cis-aconitate by another one is expected to require significant conformational changes in the cleft from the catalytic center to the protein surface. It has not been established that the leaving and entering cA-aconitases are not, in fact, one and the... 

Recently the investigation of the structure, molecular dynamics and action mechanism of enzymes revealed that protein globules of many enzymes consist of two tightly packed knots (matrix, domains, blocks) tethered with a relatively flexible spacer. (Lumry, 1995a,b, 2002 and references herein) (See also Section 4.1). The enzyme active sites are most commonly located in a cleft between these domains. Binding of substrates and inhibitors depends on the extend of matrix contraction (Fersht, 1999). 

Neurotransmitters are removed by translocation into vesicles or destroyed in enzyme-catalysed reactions. Acetylcholine must be removed from the synaptic cleft to permit repolarization and relaxation. A high affinity acetylcholinesterase (AChE) (the true or specific AChE) catalyses the hydrolysis of acetylcholine to acetate and choline. A plasma AChE (pseudo-AChE or non-specific AChE) also hydrolyses acetylcholine. A variety of plant-derived substances inhibit AChE and there is considerable interest in AChE inhibitors as potential therapies for cognition enhancement and for Alzheimer s disease. Organophosphorous compounds alkylate an active site serine on AChE and the AChE inhibition by this mechanism is the basis for the use of such compounds as insecticides (and unfortunately also as chemical warfare agents). Other synthetics with insecticidal and medical applications carbamoylate and thus inactivate AChE (Table 6.4). 

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