Effect of (auto)phosphorylation on the kinetic behavior of the Ca2+/calmodulin-dependent protein kinase II from horse brain.

The research focuses on understanding how the process of (auto)phosphorylation affects the enzymatic functions of the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) extracted from horse’s brain.

Experimental Procedure

• The study begins with the extraction and purification of CaM kinase II from horse brain.
• The kinetic behavior of this enzyme is examined as a function of two variables: ATP (Adenosine triphosphate) and a synthetic substrate named syntide-2.
• Both phosphorylated (the process where a phosphate group is added to a molecule) and dephosphorylated (the process of removing a phosphate group from a molecule) states of the enzyme are considered in the study.

Findings and Interpretation

• The researchers found that both phospho- and dephospho- forms of the enzyme obey a bi-bi random mechanism – an elementary bi-substrate enzymatic mechanism where two substrates randomly bind to the enzyme before the two products are randomly released.
• The enzyme’s affinity towards ATP and syntide-2 (measured as the Michaelis constant, K(M)) was determined to be 80 and 30 microM, respectively. This means that lower the K(M) value, higher is the enzyme’s affinity for that particular substrate.
• It was observed that the maximum achievable reaction yield decreases by 50% when the enzyme is subjected to (auto)phosphorylation. This means that the process of adding phosphate groups decreases the overall enzymatic efficiency.
• Interestingly, when the enzyme is phosphorylated, it becomes totally independent of Ca2+. This indicates that Ca2+ is no longer required for the enzymatic reaction to occur once the enzyme is phosphorylated.