Asp177 in C4 domain of mouse sphingosine kinase 1a is important for the sphingosine recognition.
Sphingosine kinase (SK) is the enzyme that catalyzes the formation of sphingosine 1-phosphate (S1P). Although diverse biological functions have been reported for SK, its recognition site for its substrate sphingosine (Sph) is still unclear. We constructed various mutants of mouse sphingosine kinase 1a (mSK1a), carrying mutations in the C4 domain, which we had expected to encompass the Sph-binding site. We analyzed the influence of these mutations on the SK activity and substrate kinetics. One mutation, Asp177-->Asn177, caused a dramatic decrease in SK activity (to approximately 6% of wild type) and an increase in the Km value for Sph (10.1-->108 microM), with no change in the affinity for ATP. This result suggests that the C4 domain, especially the Asp177, is involved in the specific recognition of Sph. In this report, we are able, for the first time, to provide an account of the Sph-binding site of SK.
Yokota
Shinji
S
Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan.
Taniguchi
Yuki
Y
Kihara
Akio
A
Mitsutake
Susumu
S
Igarashi
Yasuyuki
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013110:Sphingosine / Q000378:metabolism; D000099157:Sphingosine Kinase
D000255:Adenosine Triphosphate; D001224:Aspartic Acid; D002147:Calmodulin; D007527:Isoenzymes; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase