Thermal stability of Artemia HGPRT: effect of substrates on inactivation kinetics.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, E.C.2.4.2.8) from Artemia cysts exhibits maximum activity at 70 degrees C. Its thermal stability has been examined following enzymatic activity as a function of temperature. Cold-induced renaturation experiments of samples heated at increasing temperatures showed that reversibility of thermal inactivation depends on the incubation time and final temperature. Prolonged incubation of the thermoinactivated enzyme at 0 degree C did not afford any further increase of the catalytic activity at 37 degrees C. The complex substrate PRPP:Mg protects HGPRT from thermal inactivation. However, incubations with hypoxanthine rendered a less thermostable enzyme at any temperature tested. The irreversible inactivation of HGPRT proceeds in two exponential steps. The analysis of the apparent rate constants for the fast and the slow phases, lambda 1 and lambda 2 as per the Lumry and Eyring model suggests the existence of more than three states in the thermal denaturation pathway of the free enzyme. In the presence of PRPP:Mg the irreversible process follows a single exponential and proceeds very slowly below 70 degrees C. PRPP:Mg also protects the enzyme from inactivation by NEM and pCMB, suggesting that -SH groups may be in the vicinity of the active site.
Montero
C
C
Instituto de Investigaciones Biomedicas del Consejo Superior de Investigaciones Cientificas, (C.S.I.C), Madrid, Spain.
Llorente
P
P
Argomaniz
L
L
Menendez
M
M
D000818:Animals; D001156:Artemia / Q000201:enzymology*; D001665:Binding Sites; D004795:Enzyme Stability; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D010754:Phosphoribosyl Pyrophosphate; D011489:Protein Denaturation; D017510:Protein Folding; D013379:Substrate Specificity; D013696:Temperature
D046911:Macromolecular Substances; D010754:Phosphoribosyl Pyrophosphate; D007041:Hypoxanthine Phosphoribosyltransferase