Blood group B galactosyltransferase: insights into substrate binding from NMR experiments.
The biosynthesis of human blood group B antigens is accomplished by a highly specific galactosyltransferase (GTB). On the basis of NMR experiments, we propose a "molecular tweezers mechanism" that accounts for the exquisite stereoselectivity of donor substrate selection. Transferred NOE experiments for the first time reveal the bioactive conformation of the donor substrate UDP-galactose (UDP-Gal) and of its enzymatically inactive analogue, UDP-glucose (UDP-Glc). Both bind to GTB in a folded conformation that is sparsely populated in solution, whereas acceptor ligands bind in a conformation that predominates in solution. The bound conformations of UDP-Gal and UDP-Glc are identical within experimental error. Therefore, GTB must discriminate between the two activated sugars on the basis of a hitherto unknown transition state that can only be formed in the case of UDP-Gal. A full relaxation and exchange matrix analysis of STD NMR experiments reveals that acceptor substrates dissociate significantly faster (k(off) > 100 Hz) from the binding pocket than donor substrates (k(off) approximately 10 Hz). STD NMR experiments also directly show that proper recognition of the hexopyranose rings of the UDP sugars requires bivalent metal cations. At the same time, this analysis furnishes the complete three-dimensional structure of the enzyme with its bound donor substrate UDP-Gal on the basis of a prior crystal structure analysis. We propose that, upon acceptor binding, GTB uses the Asp 302 and Glu 303 side chains as "molecular tweezers" to promote bound UDP-Gal but not UDP-Glc into a transition state that leads to product formation.
Angulo
Jesus
J
Institute of Chemistry, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany.
Langpap
Brigitte
B
Blume
Astrid
A
Biet
Thorsten
T
Meyer
Bernd
B
Krishna
N Rama
NR
Peters
Hannelore
H
Palcic
Monica M
MM
Peters
Thomas
T
D000017:ABO Blood-Group System; D001665:Binding Sites; D001789:Blood Group Antigens / Q000096:biosynthesis / Q000097:blood* / Q000737:chemistry; D005700:Galactosyltransferases / Q000097:blood* / Q000737:chemistry; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D013379:Substrate Specificity; D013997:Time Factors; D014531:Uridine Diphosphate Galactose / Q000737:chemistry / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000378:metabolism
D000017:ABO Blood-Group System; D001789:Blood Group Antigens; D008024:Ligands; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; C023155:blood-group-substance alpha-D-galactosyltransferase; D014532:Uridine Diphosphate Glucose