The transcriptional induction of the GAL genes of Saccharomyces cerevisiae occurs when galactose and ATP interact with Gal3p. This protein-small molecule complex associates with Gal80p to relieve its inhibitory effect on the transcriptional activator Gal4p. Gal3p shares a high degree of sequence homology to galactokinase, Gal1p, but does not itself possess galactokinase activity. By constructing chimeric proteins in which regions of the GAL1 gene are inserted into the GAL3 coding sequence, we have been able to impart galactokinase activity upon Gal3p as judged in vivo and in vitro. Remarkably, the insertion of just two amino acids from Gal1p into the corresponding region of Gal3p confers galactokinase activity onto the resultant protein. The chimeric protein, termed Gal3p؉SA, retains its ability to efficiently induce the GAL genes. Kinetic analysis of Gal3p؉SA reveals that the K m for galactose is similar to that of Gal1p, but the K m for ATP is increased. The chimeric enzyme was found to have a decreased turnover number in comparison to Gal1p. These results are discussed in terms of both the mechanism of galactokinase function and that of transcriptional induction.T he yeast Saccharomyces cerevisiae utilizes galactose by means of the enzymes of the Leloir pathway. When yeast are grown in the absence of galactose, the genes encoding the enzymes of the pathway (the GAL genes) are transcriptionally inert (reviewed in refs. 1-3). If the cells are switched to medium in which galactose is the sole carbon source, then the GAL genes are rapidly induced and transcribed at high levels (4). The induction of the GAL genes is controlled by the interplay of three proteins-a transcriptional activator, Gal4p, a repressor, Gal80p, and an inducer, Gal3p. Induction appears to occur as a result of a galactose-and ATP-dependent interaction between . This association results in the formation of a transcriptionally active Gal4p-Gal80p-Gal3p complex (9). It has been suggested that the association of Gal3p with Gal80p results in the movement of Gal80p from the activation domain of Gal4p to a different part of the protein (10). The location of this second site of Gal80p interaction on Gal4p remains unclear.The first step of the Leloir pathway is the conversion of galactose to galactose-1-phosphate by galactokinase by Gal1p (11), the product of the GAL1 gene. Gal1p and Gal3p are highly homologous proteins (73% identity and 92% homology at the amino acid level). Unlike Gal1p, Gal3p does not possess a galactokinase activity (12). Gal1p is bifunctional in that it has galactokinase activity and is able to induce the expression of the GAL genes both in vivo (13) and in vitro (9), although approximately 40-fold less efficiently than Gal3p (9). Galactokinases are relatively well conserved throughout nature. For instance, Gal1p and Escherichia coli galactokinase, galK, share 50% amino acid homology. Galactokinases are, however, extremely highly conserved in five regions (Fig. 1). The functions of these regions have not been defined experimentall...

The cDNA and derived amino acid sequence of human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database. This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases. It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi.

Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase and Ap4A pyrophosphohydrolase activities have been purified from extracts of the green alga Scenedesmus obliquus. Both activities were also detected in Scenedesmus brasiliensis, Scenedesmus quadricauda and in Chlorella vulgaris. This is the first time that both types of enzyme have been detected in the same species. The Ap4A phosphorylase has a molecular mass of 46-48 kDa, a broad pH optimum between 7.5 and 9.5, and requires a divalent ion for activity (Mg2+ > Co2+ > Ca2+ = Mn2+ = Cd2+ > Zn2+). It degrades substrates with at least four phosphate groups and always produces a nucleoside 5'-diphosphate product. The Km values for Ap4A and Pi are 5.3 microM and 160 microM, respectively, and kcat. = 1.8 s-1. Arsenate, vanadate, molybdate, chromate and tungstate can substitute for phosphate. The enzyme also catalyses Ap4A synthesis (Keq. = [Ap4A] [Pi]/[ATP][ADP] = 9 x 10(-4)) and ADP arsenolysis. The Ap4A hydrolase has a molecular mass of 26-28 kDa, an alkaline pH optimum of 8.8-9.8, and prefers Zn2+ as the stimulatory ion (Zn2+ > Mg2+ > Mn2+ > Co2+ > Cd2+). It degrades substrates with at least four phosphate groups, having a slight preference for Ap5A, and always produces a nucleoside 5'-triphosphate product. The Km value for Ap4A is 6.6 microM and kcat. = 1.3 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 0.67 microM) and non-competitively by fluoride (Ki = 150 microM). A 50-54 kDa dinucleoside 5',5'''-P1,P3-triphosphate (Ap3A) pyrophosphohydrolase was also detected in S. obliquus, S. quadricauda and C. vulgaris. The corresponding enzyme in S. brasiliensis (> 100 kDa) may be a dimer

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