Related Experiment Videos
Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation
1Department of Pharmaceutical Chemistry and Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Biochemistry
|August 10, 2000
Summary
S-adenosylhomocysteine hydrolase undergoes a conformational change from an open to a closed state upon substrate binding. This domain closure stabilizes the active site before substrate oxidation, as revealed by rotational dynamics studies.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Crystal structures reveal distinct open and closed conformations of S-adenosylhomocysteine (AdoHcy) hydrolase in its substrate-free (NAD+) and substrate-bound (NADH) forms.
- These structural differences highlight significant variations in the spatial arrangement of catalytic and NAD+ binding domains.
Purpose of the Study:
- To investigate whether domain closure in AdoHcy hydrolase is triggered by substrate binding or subsequent oxidation.
- To elucidate the dynamic mechanisms governing the enzyme's conformational transitions.
Main Methods:
- Utilized spectroscopic probes covalently attached to Cys(113) and Cys(421) to measure rotational dynamics of catalytic and carboxyl-terminal domains.
- Analyzed changes in protein hydrodynamic volume following substrate (adenosine, neplanocin A) or nonsubstrate (3'-deoxyadenosine) binding.
Main Results:
- An independent motion of the catalytic domain, indicative of a flexible hinge, was observed prior to substrate binding.
- Substrate or nonsubstrate binding abolished this independent catalytic domain motion.
- A significant decrease in the enzyme's hydrodynamic volume was detected post-substrate binding, consistent with domain closure.
Conclusions:
- Substrate binding, rather than oxidation, induces the closure of the catalytic and NAD+ binding domains in AdoHcy hydrolase.
- This domain stabilization forms a closed active site through substrate interactions before oxidation occurs.