Substrate specificity of rat brain ceramidase.
In this study, the substrate specificity of a newly identified rat brain ceramidase (CDase) was investigated. To this end, the major functional groups and stereochemistry of ceramide (Cer) were evaluated for their influence on the hydrolysis of substrate by this CDase. The results showed that, of the four possible stereoisomers of Cer, only the natural d-e-C(18)-Cer isomer was used as substrate (K(m) of 1.1 mol% and V(max) of 5 micromol/min/mg). Removal of the 4-5 trans double bond to generate dihydroceramide decreased the affinity of the enzyme toward its substrate by around 90%, whereas changing the configuration of the double bond from the natural trans configuration into cis or introduction of a hydroxyl group (phytoceramide) resulted in loss of hydrolysis. Shortening the chain length of the sphingosine backbone resulted in decreased affinity. Methylation of either the primary or the secondary hydroxyl groups resulted in loss of activity. Results also indicated that Cer species that harbor long saturated or monounsaturated fatty acyl chains are preferred substrates of the enzyme. alpha-Hydroxylated Cer demonstrated considerably higher affinity, indicating a preference of the enzyme to those Cer molecular species. These results disclose a very high specificity of nonlysosomal CDase for its substrate, Cer.
El Bawab
Samer
S
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.
Usta
Julnar
J
Roddy
Patrick
P
Szulc
Zdzislaw M
ZM
Bielawska
Alicja
A
Hannun
Yusuf A
YA
D000577:Amides / Q000737:chemistry; D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D055572:Ceramidases; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013110:Sphingosine / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000502:physiology; D000099252:Fatty Acid Amide Hydrolases
D000577:Amides; D000581:Amidohydrolases; D055572:Ceramidases; D002518:Ceramides; D013110:Sphingosine; D000099252:Fatty Acid Amide Hydrolases; C109343:dihydroceramide