Kinetics of ATP and Hexokinase PII Binding Using TNP-ATP as a Probe in the Presence and Absence of Glucose

Brinser, Molly

Kinetics of ATP and Hexokinase PII Binding Using TNP-ATP as a Probe in the Presence and Absence of Glucose

Persistent URL

http://hdl.handle.net/10456/41497

Author(s)

Brinser, Molly

Date Issued

April 2016

Abstract

The glucose binding site in hexokinase is well known, but the ATP binding site is not as well understood. There is controversy over whether ATP prefers to have glucose present in order for binding to occur. Hexokinase and ATP binding was then studied in the presence and absence of glucose using TNP-ATP as an ATP analog. The kinetics of the association and dissociation reactions between hexokinase and TNP-ATP were monitored using absorbance stopped-flow. The association reaction in the absence of glucose (kon,n) between hexokinase and TNP-ATP was second order and the rate constant was approximately 4000 1/sec. The dissociation reaction in the absence of glucose (koff,n) between hexokinase and TNP-ATP was first order and the rate constant was 9.87 1/μMsec. The association reaction in the presence of glucose (kon,g) between hexokinase and TNP-ATP was presumed to be second order with the rate constant > 9.87 1/μMsec. The dissociation reaction in the presence of glucose (koff,g) between hexokinase and TNP-ATP was presumed to be first order with the rate constant > 4000/s. The fact that the rate constants for the glucose reactions were too large to measure, suggests that ATP binding prefers for glucose to be present.

Major

Chemistry

First Reader(s)

Deckert, Alice A.