The Effect on Activity of Inducible Nitric Oxide Synthase by a Site III Calmodulin Mutant

Abstract

Nitric oxide (-NO) has many physiological roles in the human body. From fighting infection to controlling blood pressure -NO is an important physiological molecule. However, the line between being beneficial and being a danger to the human body is thin. -NO in combination with oxygen radicals can form peroxynitrites, which interact and damage DNA, lipids, and proteins and lead to cell death. Any effort at controlling the formation of excess -NO requires an indepth understanding of NOS. The NOS enzyme catalyzes the conversion of Larginine to L-citrulline and -NO. There are three different enzyme isoforms of NOS found in the human body, and all catalyze this same reaction. Recent work by Dr. Stevens-Truss et al. have focused on the relationship between the NOS isoforms and the calmodulin (CaM) subunit, which is needed by NOS to efficiently pass electrons from the reductase to the heme domain of the enzyme. In this report a CaM mutant called B3Q was investigated, and the effect it had on the specific activity of iNOS (an NOS isoform found in human macrophages) was examined. However, first a cost effective and reliable purification protocol for isolating the iNOS/B3Q complex was established. Exploring any type of treatment relating to manipulating the ability of an enzyme to function requires an in-depth understanding, and in this report the successful isolation of the iNOS/B3Q complex has resulted in the discovery that a B3Q CaM mutant reduces iNOS specific activity to 43% of maximal. Based upon the results a structural hypothesis of the interaction between iNOS and CaM is presented.