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Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B 2 ) to form FMN, an obligatory step in vitamin B 2 utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel β barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3 10 helix, and a reverse turn followed by a short β strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.

In mammals, melatonin activates melatonin MT 1 and MT 2 receptors. Using site-directed mutagenesis and chemical modification, we investigated the role of conserved cysteines in ligand binding. Dithiothreitol inhibited 2-[ 125 I]iodomelatonin binding to the FLAG-tagged human melatonin MT 2 receptor without affecting ligand affinity. Alanine substitution of Cys 113 or Cys 190 resulted in a loss of specific 2-[ 125 I]iodomelatonin binding, without altering cell surface receptor expression. This suggests that a putative disulfide bond linking Cys 113 and Cys 190 is essential to maintain a proper human melatonin MT 2 receptor conformation for melatonin binding. N -ethylmaleimide alkylation of cysteines inhibited 2-[ 125 I]iodomelatonin binding, decreasing both ligand affinity and receptor density. Alkylation of Cys 140 contributes to changes in ligand affinity, while alkylation of Cys 143 and Cys 219 reduced binding capacity. We suggest that a disulfide bridge is important for the proper structural conformation of the human melatonin MT 2 receptor to bind melatonin. Cysteines located in receptor regions near the ligand binding site and/or G protein coupling region are involved in N -ethylmaleimide-induced changes in affinity and receptor density.

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase (PDE2) is the second most abundant of this class of enzymes in platelets. PDE2 probably plays an important role in the regulation of elevated intracellular concentrations of cAMP and cGMP in platelets inhibited by prostacyclin and/or nitric oxide. The cAMP and cGMP PDEs have catalytic domains with 28–40% identity, but vary in their substrate specificity and affinity. As a first step toward the goal of identifying important amino acids in the substrate binding site pocket, we have employed the affinity analog 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine-3′5′ cyclic monophosphate (8-BDB-TcAMP) to inactivate PDE2 and observe the pattern of protection by substrates and their products. Incubation of purified platelet PDE2 with 8-BDB-TcAMP (2–10 mM) resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.013 min−1 mM−1. Both substrates, cAMP and cGMP, as well as the products of hydrolysis by PDE2, AMP and GMP, exhibited concentration-dependent protection against inhibition by 8-BDB-TcAMP, but no protection was noted with ADP or ATP, which are not hydrolyzed by the enzyme. This compound, 8-BDB-TcAMP, and similar affinity reagents should prove useful in delineating amino acids in the active site of PDE2.