Asp48 E(-1) D(-2) H (c) ( To identify the critical enzyme resi- due:substrate interaction and determinant of sub- strate specificity, steady-state kinetic studies have D(-4) been carried out by site-specific mutagenesis of the enzyme and by systematically changing amino acid residues in the 11-amino acid phosphotyrosine con- taining substrate that simulate residues 988 - 998 of the epidermal growth factor receptor (EGFR 988-998). The table below compares the kinetic parameters of the wild type enzyme with phosphotyrosine pep- tides in which amino acid residues are substituted. In the adjacent diagram the structural interactions between active site residues are illustrated for the first 6 residues of the substrate. Identify which sub- strate residue is most sensitive to substitution and justify your conclusion on the basis of the kinetic parameters. Is the decrease in Kcat/ KM due to loss of catalytic reactivity or due to suboptimal position- ing of the phospho-Tyr residue in the active site? A(-3) Tyr46 Ser216 Arg47 Asp181 Substrate DADEpYLIPQQG DADAP YLIPQQG DAAEpYLIPQQG AAAAP YLIPQQG kcat 5-1 44.6 ± 1.8 39.8 ± 0.32 35.3 ± 0.22 34.7 ± 0.25 PTP1B Km Y(0) L(+1) Gln262 Phe 182 kcat/Km им 3.9 ± 0.9 13.7 ± 0.46 6.6 ± 0.22 52.7 ± 0.7 10-7 × (s¯¹ M¯¹) 1.1 ± 0.25 0.29 ± 0.01 0.53 ± 0.02 0.066 ± 0.001

Mutation identity- GLY Outline the effects the mutation of cysteine with GLY on the 3D structure and function of  the 3GRS glucathione reducatse protein.

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Kk.31.

13. Amino acid analysis of a seven residue long peptide gave Asp Leu Arg Met | Phe Met | Phe | Tyr (i) The PTH amino acid derivative released by Edman degradation was Tyr-PTH (ii) Trypsin treatment had no effect (iii) Cyanogen bromide treatment yielded a dipeptide, a tetrapeptide, and free Arg (iv) Brief chymotrypsin treatment yielded several products, including a dipeptide and a tetrapeptide. The amino acid composition of the tetrapeptide was Leu, Arg, and Met What is the amino acid sequence of this seven residue long peptide, and explain how you reached that conclusion?