Identification of amino-acid residues linked to different properties of phosphoribosylpyrophosphate synthetase isoforms I and II.
Biochim Biophys Acta
; 1207(1): 126-33, 1994 Jul 20.
Article
in En
| MEDLINE
| ID: mdl-8043603
ABSTRACT
The catalytic subunit of rat liver phosphoribosylpyrophosphate synthetase is composed of two isoforms, PRS I and PRS II. The amino-acid sequences differ only by 13 residues, out of which two Lys residues of PRS I at positions 4 and 152 give net additional positive charges to PRS I. Previous work has shown that PRS I is more sensitive to inhibition by ADP and GDP and more stable to heat treatment than is PRS II. To identify amino-acid residues responsible for the different properties, five chimeric enzymes between rat PRS I and PRS II and two mutated enzymes with a single point mutation at position 152 were constructed; these enzymes were produced in Escherichia coli. Changing Lys-4 of PRS I to Val, together with Ile-5 to Leu, completely abolished sensitivity to GDP inhibition of PRS I, indicating that Lys-4 in PRS I is critical for GDP inhibition. The substitutions at position 152 had little effect on GDP inhibition. Characterization of the chimeric enzymes revealed that residues between residues 54-110 and 229-317, namely, Val-55 and/or Ala-81, and Arg-242 and/or Cys-264 of PRS I also contribute to the strong GDP inhibition. Lys-4 was also important for the strong ADP inhibition of PRS I. Regarding the physical properties, chimeric enzymes bearing residues 12-53 of PRS I were stable at 49 degrees C and with digestion with papain and proteinase K. Our observations suggest that Lys-17, Ile-18, and/or Cys-40 of PRS I contribute to stability of the enzyme.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Ribose-Phosphate Pyrophosphokinase
/
Amino Acids
/
Isoenzymes
Type of study:
Diagnostic_studies
Language:
En
Journal:
Biochim Biophys Acta
Year:
1994
Type:
Article
Affiliation country:
Japan