Biological Background and Chemical Mechanism

Living organisms have developed features to ensure their existence in a wide variety of environments on Earth. While details of these features may be unique to particular conditions or species, two minimum requirements are the ability to reproduce and carry out regulated metabolic processes. A common theme in metabolic processes is the synthesis of complex and diverse molecules from a limited number of precursors. Amino acids are not only the building blocks of proteins and peptides, they are also important precursors in the biosynthesis of purines, pyrimidines, and other biomolecules. Additionally, amino acid biosynthesis is an ancient and fundamental process, and these metabolic pathways are represented in a diversity of organisms spanning all three domains of life. Regulated production of histidine depends on the complex interplay between nine catalytic active sites located on 6-8 polypeptide chains, depending on the organism. High resolution crystal structures of several of the enzymes regulating this vital pathway are now available [15,16]. Of particular interest is the fifth step of the metabolic pathway, where a protein complex known as hisH-hisF forms a key branch point. At this step, the formation of two products is catalyzed by the heterodimeric enzyme complex, imidazole glycerol phosphate synthase (IGPS), which consists of hisH, a class-I glutamine amidotransferase, and hisF, a synthase subunit that catalyzes a cyclase reaction. One product, imidazole glycerol phosphate (ImGP), is further used in histidine biosynthesis, and the other, 5-aminoimidazole-4-carboxamide ribotide (AICAR), initiates de novo synthesis of purines (see [17,18] and references therein).

Figure 1: HisH mechanism for glutaminase reaction; we will model the system after step 2 of this mechanism (middle, right).

Characteristic of the superfamily to which it belongs, hisH has a strictly conserved catalytic triad active site: CYS84, HIS178, GLU180. The cysteine covalently binds glutamine, and the histidine, initially protonated, donates a proton to the amide group of glutamine to produce ammonia and glutamate. The conserved chemical mechanism for another enzyme (Carbamoyl Phosphate Synthetase) belonging to this superfamily is depicted below [19]. Subsequent steps allow the release of glutamate and the reprotonation of the active site histidine (HIS178). The molecule of ammonia then diffuses roughly 10 angstroms across the interface of the two proteins, enters the alpha-beta barrel of hisF through a presently unknown mechanism, and is transported through the barrel of hisF to the active site located at the C-terminal end of the barrel where it is incorporated into the next substrate. This exercise will lead you through the modelling of the hisH protein. We will investigate the catalytic triad that comprises its active site and determine the correct protonation states of all functionally important residues. After the cysteine has covalently bound glutamine, we are presented with a non-standard amino acid to simulate. It is our task to develop a set of force field parameters for the novel residue for use in MD simulations.