Functional characterization enzymes

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

D. desulfuricans is able to grow on nitrate, inducing two enzymes that responsible for the steps of conversion of nitrate to nitrite (nitrate reductase-NAP), which is an iron-sulfur Mo-containing enzyme, and that for conversion of nitrite to ammonia (nitrite reduc-tase-NIR), which is a heme-containing enzyme. Nitrate reductase from D. desulfuricans is the only characterized enzyme isolated from a sulfate reducer that has this function. The enzyme is a monomer of 74 kDa and contains two MGD bound to a molybdenum and one [4Fe-4S] center (228, 229) in a single polypeptide chain of 753 amino acids. FXAFS data on the native nitrate reductase show that besides the two pterins coordinated to the molybdenum, there is a cysteine and a nonsulfur ligand, probably a Mo-OH (G. N. George, personal communication). 

These examples highlight the utility of competitive ABPP and CC-ABPP to identify the targets and selectivity of existing small molecules, to identify novel covalent enzyme inhibitors, and to characterize enzymes of unknown function. 

Liger D, Graille M, Zhou CZ, Leulliot N, Quevillon-Chemel S, Blondeau K, Janin J, van Tilbeurgh H (2004) Crystal structure and functional characterization of yeast YLROllwp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities. J Biol... 

LAUDERT, D., PFANNSCHMIDT, U., LOTTSPEICH, F HOLLANDER-CZYTKO, H., WEILER, E.W., Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP74) the first enzyme for the octadecanoid pathway to jasmonates, Plant Mol. Biol., 1996,31,323-335. 

The GRK family consists of seven well-characterized enzymes. These enzymes are distinguished by (1) the structural homology within the family (2) the specific amino acid sequences that a given GRK can phosphorylate (3) enzyme kinetics (169,181) and (4) GPCR disease phenotypes that are often manifested by dysregula-tion of GRK activity. Gain-of-function GPCR mutations are frequently found to be constitutively phosphorylated. Conversely, inadequate receptor desensitization and sequestration often result. 

Frydman A, Weisshaus O, Bar-Peled M, Huhman D, Summer L, Matin F, Lewinsohn E, Fluhr R, Gressel J, Eyal Y (2004) Citrus fruit bitter flavors isolation and functional characterization of the gene Cml,2RhaT encoding a 1,2rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of Citrus. Plant J 40 88-100... 

A further advantage, as described by Thomas et al. [19], is the possibility of protein identification that follows the functional characterization of the enzyme. The activity of an enzyme is initially determined by following the substrate consumption and product formation in the first assay (Fig. 8.10). Since no matrix components are present in the sample spot, the immobilized enzyme is then directly... 

Fig. 8.10 Sequential functional characterization and structural identification of an enzyme. Initially, information about the activity is obtained by assessing substrate consumption and product formation. Afterwards, the enzyme is digested on the plate, and the formed peptide fragments (F1-F4) are determined by means of mass spectrometry.

The structure and function of enzymes is determined by both the amino acid sequence and the surrounding solvent. The overall stability of proteins is characterized by a subtle balance of into- and inter-molecular interactions. The basic principle of the structure (and of the stability) of the proteins is related to the nature of its normal enviromnent for (water) soluble globular proteins this is the minimization of the hydrophobic surface area, whereas the contrary is the case for membrane proteins (Jaenicke, 1991). 

Figure 10.4 Examples of covalent capture methods. (A) Covalent modification of native cysteines has been shown to modulate ion-channel activity and in the case of enzymes lead to allosteric inhibition. These findings can be important for structural-functional characterization or as starting points for drug discovery. (B) Reversible covalent capture using imine chemistry.

Staub I, Sieber SA (2009) P-Lactam probes as selective chemical-proteomic tools for the identification and functional characterization of resistance associated enzymes in MRSA. J Am Chem Soc 131 6271-6276... 

The identification of changes in the metabolome can help characterize the biochemical functions of enzymes in the proteome [5, 24]. Not all metabolic changes, however, provide easy readouts. Therefore, more sophisticated analytical methods have been developed to detect and quantify changes in metabolome. The application of modem analytical tools, such as nuclear magnetic resonance (NMR) [19] and mass spectrometry (MS) [5, 24], are the primary tools of metabolomics researchers. In particular, MS has found increased usage as mass spectrometers have improved their sensitivity and mass resolution. 

M. Lindenmeier, V. Faist, and T. Hofmann, Structural and functional characterization of pronyl-lysine, a novel protein modification in bread crust melanoidins showing in vitro antioxidative and Phase Eli enzyme modulating activity, J. Agric. Food Chem., 2002, 50, 6997-7006. 

Proteins constitute the largest portion of living matter in all types of cells. They serve as structural elements in cells and tissues, show specific catalytic activity, function as enzymes, and are found in cell membranes. Most IR and classical Raman studies focus on the characterization of protein secondary structures by using sets of absorption bands and diffusion Raman lines reflecting especially the motions of peptide groups (Sec. 

---