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Yeast protein geranylgeranyltransferase type-I: steady-state kinetics and substrate binding
1Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Biochemistry
|April 15, 1997
Summary
Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes protein alkylation. Kinetic studies reveal geranylgeranyl diphosphate (GGPP) binds before the peptide substrate, indicating an ordered reaction mechanism.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Protein geranylgeranyltransferase type-I (PGGTase-I) is crucial for post-translational modification.
- It catalyzes the alkylation of proteins with a C-terminal CaaX motif.
Purpose of the Study:
- To elucidate the kinetic mechanism of PGGTase-I.
- To determine the substrate binding order for PGGTase-I.
Main Methods:
- Continuous fluorescence-based kinetic assay using dansyl-GCIIL.
- Enzyme kinetics with varying substrate concentrations.
- Inhibitor studies using dead-end inhibitors.
Main Results:
- Determined kinetic constants: k(cat) = 0.34 s(-1), K(M)(GGPP) = 0.86 microM, K(M)(dansyl-GCIIL) = 1.6 microM.
- Inhibitor studies indicated GGPP binds before the peptide substrate.
- Dissociation constant for the PGGTase-I x GGPP complex (K(D)) was 120 nM.
Conclusions:
- PGGTase-I follows an ordered sequential binding mechanism.
- Geranylgeranyl diphosphate (GGPP) is the initial substrate to bind.
- This understanding is vital for drug development targeting protein prenylation.