Mutational analysis of the important amino acids in the catalytic triad of serine proteases showed that replacing Asp102 or modifying His57 with amethyl group decreased the reaction rate about 5000-fold. However,mutating Ser195 decreased the reaction rate a millionfold. Discuss why mutating Ser had a larger impact than the other members of the catalytic triad.
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Mutational analysis of the important amino acids in the catalytic triad of serine proteases showed that replacing Asp102 or modifying His57 with amethyl group decreased the reaction rate about 5000-fold. However,mutating Ser195 decreased the reaction rate a millionfold. Discuss why mutating Ser had a larger impact than the other members of the catalytic
triad.
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- A Leu →Ala mutation at a site buried in the core of the enzyme lysozymeis found to be destabilizing. Explain the observed effect of this mutationon lysozyme stability by predicting how enthalpy (ΔH°), conformationalentropy (ΔS°peptide), and the hydrophobic effect (ΔS°solvent) are expected to change for the mutant compared to wild-type lysozyme. Explain how ΔG°for unfolding is affected by your predicted changes in enthalpy or entropy.(c) On the right is a diagram of the ac tive site of E. coli aspartate aminotrans- ferase illustrating the cofactor pyridoxal phosphate (labeled PLP) with the dicar- boxylic acid maleate (labeled MAL) bound in the active site. The structural formula of maleate is shown on the right. Am 194 MAL Arg292 Arg386 Ilx17 Lauf 'coo- H get H Coo- Maleate (c1) Draw the structure of L-aspartate and draw a border around the atoms in the amino acid that maleate simulates. (c2) Identify the active site residues that make hydrogen bonds and electrostatic interac- tions with the oxygen atoms of the carboxylate groups of maleate in the diagram above. Identify the carboxylate groups according to the numbering in the diagram of maleate above. Indicate the hydrogen donor groups of the active site residues. (C3) Compare and draw the structures of L-Arg and L-Lys. On the basis of the diagram why does replacement of an arginine for a lysine have an effect on substrate binding to AspAT? (c4) Of the mutant…An active site of a hypothetical serine protease with a peptide substrate bound is shown below: This serine protease has 3 specificity pockets (S1, S2, S1') as shown in the figure above. S1 pocket has a glutamic acid in the bottom, the S2 pocket is small and hydrophobic, and the S1' pocket is deep and hydrophobic. Based on this information you can conclude that R1 is most likely side chain of a. Asp b. Lys c. Lle d. Gly e. Phe
- Replacing Glu-165 with alanine has the most significant impact on the catalytic activity of all the mutations examined. Does this observation agree with your predictions about the roles of the various active site residues, informed by the computational mutagenesis?Mutants of Neurospora crassa that lack carbamoyl phosphate synthetase I (CPS I) require arginine in the medium in order to grow, whereas mutants that lack carbamoyl-phosphate synthetase II (CPS II) require a pyrimidine, such as uracil. A priori, one would expect the active CPS II in the arginine mutants to provide sufficient carbamoyl phosphate for arginine synthesis, and the active CPS I in the pyrimidine mutants to "feed" the pyrimidine pathway. Explain these observations.The objective is to study a novel protease P isolated from the digestive tract of an Amazonian insect. This protease can exist into two forms Pi and Pa which have identical amino acid sequences (both of 80 kDa). However, only Pa shows proteolytic activity. To better understand the activation mode of Pi (inactive form) in Pa (active form), the following experiment was done using DIPF. DIPF (diisopropylphosphofluoridate) is a well-known irreversible inhibitor of serine proteases. It reacts with the catalytic serine residue of the active site of proteases as shown below: Enzyme -CH₂OH + CH(CH3)2 O F-P=0 O CH(CH3)2 Diisopropylphospho- fluoridate (DIPF) Enzyme -CH,—O CH(CH3)2 O <=0 O CH(CH3)2 DIP-Enzyme Both proteases Pa and P₁ were incubated with 32P-DIPF for 30 min at 37°C, and then dialysed to remove excess of unreacted radiolabelled reagent. The two proteases were then analyzed in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without 2-mercaptoethanol.…
- The Energy Cost of dTTP Synthesis (Integrates with Chapter 20.) Starting from HCO3, glutamine, aspartate, and ribosc-5-P, how many ATP equivalents are consumed in the synthesis of dTTP in a eukaryotic cell, assuming dihydroorotate oxidation is coupled to oxidative phosphorylation? How does this result compare with the ATP costs of purine nucleotide biosynthesis calculated in problem 2?. Mutants of Neurospora crassa that lack carbamoyl phosphate syn- thetase I (CPS I) require arginine in the medium in order to grow, whereas mutants that lack carbamoyl-phosphate synthetase II (CPS II) require a pyrimidine, such as uracil. A priori, one would expect the active CPS II in the arginine mutants to provide sufficient carbamoyl phosphate for arginine synthesis, and the active CPS I in the pyrimidine mutants to "feed" the pyrimidine pathway. Explain these observations.Can you please order the following steps involving the regeneration of ribonucleotide reductase that occurs in most animals so that it may carry out the formation of deoxyribonucleotides. (Note that not all steps are shown.) 1. Reduction of thioredoxin 2. Reduction of ribonucleotide reductase 3. Oxidation of thioredoxin reductase 4. Reduction of thioredoxin reductase
- CTP synthetase catalyzes the glutamine-dependent conversion of UTP to CTP. The enzyme is allosterically inhibited by the product, CTP. Mammalian cells defective in this allosteric inhibition are found to have a complex phenotype: They require thymidine in the growth medium, they have unbalanced nucleotide pools, and they have an elevated spontaneous mutation rate. Explain the likely basis for these observations.Escherichia coli Fpg protein is responsible for removing damaged DNA base pairs such as C8-oxoguanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA base the N-terminal proline residue. Draw the structure of PTH-derivative that is formed after Fpg is subjected to one cycle of Edman degradation.In the case of normal RNase A, Anfisen found that oxidizing the Cys residues before slowly removing the urea gave a very different result than oxidizing the Cys after slowly removing the urea. When the urea was removed first, Anfisen recovered 100% of the catalytic activity. When the urea was removed after oxidation, only about 1% of the activity (1/105) was recovered. The conclusion was that 104 of every 105 molecules was misfolded, and therefore catalytically inactive. Let's say someone raises an objection to this interpretation, and says that perhaps, instead, something chemically happens to the active site of RNase A when it is oxidized before refolding, and that the ~1% activity measured represents the residual and greatly reduced enzyme activity of all of the molecules, and the number is just coincidently similar to 1/105. (In other words, instead of 104 completely inactive molecules for every 1 completely active molecule, all molecules are equally damaged, and only function at…