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. As amino acid biosynthesis is an ancient and fundamental process, 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 [14,15,16]. 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 (IGP synthase), 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), is utilized in the de novo synthesis of purines (see [17,18] and references therein).

Figure: Putative 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 [19,20]. The conserved chemical mechanism for another enzyme (carbamoyl phosphate synthetase) belonging to this superfamily is depicted in Fig. 1 [21]. Subsequent steps allow the release of glutamate and the reprotonation of the active site histidine (HIS178). An investigation of the available crystal structures, coupled with experimental kinetic and mutagenic studies and molecular dynamics simulations, strongly suggests that the following reaction scenario: the nascent ammonia 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 [18,22].

This exercise will lead you through the steps required to create the covalently bound substrate within the active site and develop a set of parameters to use in molecular dynamics simulations.

Figure: IGP synthase, also called hisH-hisF, with bound substrates and ammonia is depicted. A molecule of glutamine binds in the active site of hisH (top, Rossman fold) and is hydrolized to form a molecule of free, reactive ammonia and glutamate. The ammonia molecule diffuses approximately 10 angstroms through the interface of the two proteins. HisF (bottom, $(\alpha /\beta )_8$ barrel) exploits its barrel structure to transport ammonia another 12 angstroms from the interface to the next active site, where it then reacts with the ribonucleotide substrate of hisF, PRFAR. PRFAR is shown docked to the C-terminal active site end of hisF.