Active site peptide

Branchini, B. R., et al. (1997). Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent. J. Biol. Cbem. 272 19359-19364. 

Stony Brook synthesized a photoreactive benzophenone containing inhibitor (39, Fig. 14) which efficiently labeled the active site of the enzyme. The photoinhibition was prevented by adding native Ras to the reaction mixture. That competition indicated that the labeling was specific at the active site. Peptide mapping of the labeled enzyme by HPLC, Edman sequencing and MALDI-MS allowed the identification of key amino acids in the substrate binding, as Asp-110 and Asp-112 in the a-subunit [126]. 

K. A. and Hollenberg, P.F. (2006) Identification of 17-alpha-ethynylestradiol-modified active site peptides and glutathione conjugates... 

He, K., Falick, A.M., Chen, B., Nilsson, F. and Correia, M.A. (1996) Identification of the heme adduct and an active site peptide modified during mechanism-based inactivation of rat liver cytochrome P450 2B1 by secobarbital. Chemical Research in Toxicology, 9 (3), 614-622. 

Kagan, H. M., and Cai, P. (1995). Isolation of active site peptides of lysyl oxidase. Methods Enzymol. 258, 122-132. 

The weakness of proteinoid function is plausible from a viewpoint of molecular evolution, and from a chemical standpoint it is due to the primitive structure of proteinoid showing a relatively simple, or an elementary, reaction such as the active site of enzyme. For example, the melanocyte stimulating activity of the proteinoid is close to the activity of the active site peptide of the hormone, MSH. 

By treating the glucose-enzyme complexes with pepsin, Mooser et al.63 later isolated and sequenced the active-site peptides from dextransucrase (GTF-S) and mutansucrase (GTF-I), each of which has a covalently linked glucose. The two peptides had nine amino acids and the following similar, but not identical, sequences ... 

TEV) protease cleavage site is inserted between the binding/reactive group and the analytical handle. Labeling of proteomes with these probes followed by (strept) avidin resin incubation yields an enriched proteome. Trypsin digest followed by filtration yields a supernatant that can be analyzed by multidimensional LC-MS/MS analysis as in the MudPIT-ABPP experiment above. In the last step, the probe-labeled active site peptides are released from the resin by cleavage with TEV protease and subjected to MudPIT analysis (Lig. 5). 

Okerberg ES, Wu J, Zhang B et al (2005) High-resolution functional proteomics by active-site peptide profiling. Proc Natl Acad Sci USA 102 4996-5001... 

Reddy, M.N., Maraganore, J.M., Meredith, S.C., Heinrikson, R.L., Kezdy, F.J. 1986. Isolation of an active-site peptide of lipoprotein lipase from bovine milk and determination of its amino acid sequence. J. Biol. Chem. 261, 9678-9683. 

Mooser G, Hefta SA, Paxton RJ, Shively JE, Lee TD (1991) Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases. J Biol Chem 266 8916-8922... 

Fidder et al. introduced an electrospray-ionization tandem mass spectrometry method for diagnosing OP exposure by measuring the mass of the OP-labeled active site peptide of human butyrylcholinesterase (Fidder et al, 2002). His starting material was 0.5 ml of human plasma from a victim of the Tokyo subway attack. The mass of the active site peptide was higher by 120 atomic mass units, compared to the mass of the unlabeled active site peptide. This added mass was exactly the added mass expected from sarin. The peptide s MS-MS fragmentation spectrum yielded the sequence of the peptide, and verified that the OP label was on serine 198, the active site serine. Examples of the MS-MS spectra from tryptic peptides of pure, OP-labeled human butyrylcholinesterase are shown in Figure 56.1. 

The Isolation and Characterization of Active Site Peptides in Lysyl Oxidase... 

B. Mass Spectral Analysis of the Active Site Peptides... 

Figure 4. Electrospray of the LO active site peptide (sample 1). The electrospray ionization mass spectrometer was scanned in noncontinuum mode over a range of m/z of 350 to 2000 at 5 s/scan (see text for details).

Vanadium(IV) complexes with two dipeptides glycyl-tyrosine and glycyl-phenylalanine (Gly-Tyr and Gly-Phe, respectively) and their oxovanadium(lV) and (V) complexes have been observed.382, 01 The complex formed with Gly-Tyr showed coordination by the peptide backbone moieties, and no interaction from the distal phenolic hydroxy group.601 A histidine-tyrosine derivatized peptide was shown to complex Viv in a pentadentate manner (120).564 The adduct between VlV and a model of the active-site peptide of protein tyrosine phosphatases has been spectroscopically characterized.602 Similar characterization of in serum suggests that protein complexes form, and that both Viv and Vv can exist as complexes with transferrin and albumin.603 ESEEM has been used to characterize the complexes of apoferritin with V02+ and suggests that the carboxylates, one water molecule, and one histidine ligand fill the coordination sites of the vanadium.604... 

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