Amino Acid Information site

Little information is available on the active site of this enzyme. In the recombinant enzyme from E. coli two cysteine residues were recognized at the positions 129 and 329, one of which seems to be at or close to the active center, as was shown by inhibition with thiol-specific reagents partly in the presence of CTP. However, exchange of these cysteine residues for other amino acids by site-directed mutagenesis did not change the enzyme activity significantly [590]. 

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

The World Wide Web has transformed the way in which we obtain and analyze published information on proteins. What only a few years ago would take days or weeks and require the use of expensive computer workstations can now be achieved in a few minutes or hours using personal computers, both PCs and Macintosh, connected to the internet. The Web contains hundreds of sites of Interest to molecular biologists, many of which are listed in Pedro s BioMolecular Research Tools (http // www.fmi.ch/biology/research tools.html). Many sites provide free access to databases that make it very easy to obtain information on structurally related proteins, the amino acid sequences of homologous proteins, relevant literature references, medical information and metabolic pathways. This development has opened up new opportunities for even non-specialists to view and manipulate a structure of interest or to carry out amino-acid sequence comparisons, and one can now rapidly obtain an overview of a particular area of molecular biology. We shall here describe some Web sites that are of interest from a structural point of view. Updated links to these sites can be found in the Introduction to Protein Structure Web site (http // WWW.ProteinStructure.com/). 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

Structural information chemical formula and stereochemistry in the case of a New Chemical Entity (NCE) or amino acid sequence and glycosylation sites in the case of a biotech product... 

The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine (DNPH Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modification produces detectable complexes, but it does not provide information as to what amino acids are involved. O-ECAT modifies carbonyl end products of protein oxidation and in addition, it can provide exact information as to the amino acids that were oxidized. Mass spec analysis of modified proteins performed after proteolysis gives the exact amino acid sequences including the sites of O-ECAT reagent modification. The same antibody that is specific for the metal chelate portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT... 

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