Hydrolysis of a slow cyclic thiophosphate substrate of RNase T1 analyzed by time-resolved crystallography.
Here we present a time-resolved crystallographic analysis of the hydrolysis of exo (Sp) guanosine 2',3'-cyclophosphorothioate by RNase T1. The use of a slow substrate and fast crystallization methods made it possible to perform the study with conventional data-collection techniques. The results support the idea that the hydrolysis reaction proceeds through a mechanism that is the inverse of the transesterification reaction. In addition, the structures provide an explanation for the differential behavior of RNase T1 towards exo- and endo-cyclic thiophosphates.
Zegers
I
I
Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Belgium. igzegers@vub.ac.be
Loris
R
R
Dehollander
G
G
Fattah Haikal
A
A
Poortmans
F
F
Steyaert
J
J
Wyns
L
L
D001236:Aspergillus oryzae / Q000201:enzymology; D001665:Binding Sites; D003198:Computer Simulation; D018360:Crystallography, X-Ray / Q000379:methods; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D013873:Thionucleotides; C001774:guanosine 2',3'-cyclophosphorothioate; D006163:Ribonuclease T1; D006152:Cyclic GMP