A laser photolysis technique has been developed to assess the quantitative significance of the delay time of hemoglobin S gelation to the pathophysiology of sickle cell disease. Changes in the saturation of hemoglobin S with carbon monoxide produced by varying the intensity of a photolytic laser beam were used to simulate changes in the saturation of oxyhemoglobin S produced by variations in oxygen pressure. The presence of polymer at steady-state saturation with carbon monoxide was determined by measurement of the kinetics of gelation after complete photodissociation. The kinetics are a very sensitive probe for polymer since small amounts of polymerized hemoglobin increase the rate of nucleation sufficiently to eliminate the delay period. First, the equilibrium gelation properties of partially photodissociated carbonmonoxyhemoglobin S were shown to be the same as partially oxygenated hemoglobin S, and the method was then used to determine the effect of saturation on the formation and disappearance of polymers in individual sickle cells. The saturation at which polymers first formed upon deoxygenation was much lower than the saturation at which polymers disappeared upon reoxygenation. The results indicate that at venous saturations with oxygen, gelation takes place in most cells at equilibrium, but is prevented from occurring in vivo because the delay times are sufficiently long that most cells return to the lungs and are reoxygenated before polymerization has begun.

The vitamin B6-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.

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, γ-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-Å resolution) and in complex with vigabatrin as well as with the close analogue γ-ethynyl-GABA (to 2.3 and 2.8 Å, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5′-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.