Faculty Collaboration Database

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation. J Biol Chem 1985 Feb 10;260(3):1535-44

Date

02/10/1985

Pubmed ID

2981860

Scopus ID

2-s2.0-0021996449 (requires institutional sign-in at Scopus site)   20 Citations

Abstract

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.

Author List

Renosto F, Seubert PA, Knudson P, Segel IH

Author

Paul Knudson MD Associate Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Adenosine Triphosphate
Drug Stability
Enzyme Activation
Enzyme Reactivators
Hot Temperature
Hydrogen-Ion Concentration
Kinetics
Molecular Weight
Penicillium
Penicillium chrysogenum
Phosphotransferases
Phosphotransferases (Alcohol Group Acceptor)