The vitamin B(6)-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and on-going research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.

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To understand why the classical two-state allosteric model of Monod, Wyman, and Changeux explains cooperative oxygen binding by hemoglobin but does not explain changes in oxygen affinity by allosteric inhibitors, we have investigated the kinetic properties of unstable conformations transiently trapped by encapsulation in silica gels. Conformational trapping reveals that after nanosecond photodissociation of carbon monoxide a large fraction of the subunits of the T quaternary structure has kinetic properties almost identical to those of subunits of the R quaternary structure. Addition of allosteric inhibitors reduces both the fraction of R-like subunits and the oxygen affinity of the T quaternary structure. These kinetic and equilibrium results are readily explained by a recently proposed generalization of the Monod-Wyman-Changeux model in which a preequilibrium between two functionally different tertiary, rather than quaternary, conformations plays the central role.T he two-state allosteric model of Monod, Wyman, and Changeux (1) represented a conceptual breakthrough in explaining the cooperative and regulated behavior of multisubunit proteins, with application to a wide range of biological systems (2-5). Monod, Wyman, and Changeux proposed that ligands control protein function by altering a preexisting equilibrium between high (R) and low (T) reactivity conformations that differ in intersubunit bonding (quaternary structure) and not by inducing conformational changes that are propagated to neighboring subunits as in a sequential model (6, 7). Enzyme activation, for example, results from preferential binding of ligands to the R quaternary structure, whereas inhibitors preferentially bind to T. However, a long-known serious deficiency in the application of the Monod-Wyman-Changeux (MWC) model to hemoglobin, the paradigm of allosteric proteins, is that inhibitors may also change oxygen (O 2 ) affinity without a change in quaternary structure (8-11). To understand this phenomenon, we have investigated the ligand binding kinetics and equilibria of hemoglobin encapsulated in silica gels in either the T or R quaternary structure (Fig. 1).Previous studies of hemoglobin encapsulated in silica gels showed greatly simplified equilibrium properties, compared with those in solution, because quaternary conformational changes are markedly slowed by the constraints of the surrounding cross-linked polymer (12-16). In sharp contrast to hemoglobin free in solution, O 2 binding to gel-encapsulated hemoglobin, like O 2 binding to the hemoglobin crystal (17-19), is noncooperative (Fig. 2). Encapsulation as the fully deoxygenated molecule traps hemoglobin in the low-affinity T quaternary structure, whereas encapsulation as the fully oxygenated molecule traps hemoglobin in the high-affinity R structure (12). Moreover, the affinity of the deoxy-encapsulated molecule is lowered by inhibitor ligands (called negative heterotropic allosteric effectors) such as protons, chloride ions, inositol hexaphosphate, and bezafibrate (Fig. 2) in the ...

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5 0 -phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the a-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.