(c) On the right is a diagram of the active site of E. coli aspartate aminotrans-ferase illustrating the cofactor pyridoxalphosphate (labeled PLP) with the dicar-boxylic acid maleate (labeled MAL)bound in the active site. The structuralformula of maleate is shown on the right.Am 194MALArg292Arg386Ilx17Lauf'coo-HgetHCoo-Maleate(c1)Draw the structure of L-aspartate and draw a border around the atoms in the aminoacid 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. Identifythe carboxylate groups according to the numbering in the diagram of maleate above. Indicate thehydrogen donor groups of the active site residues.(C3)Compare and draw the structures of L-Arg and L-Lys. On the basis of the diagramwhy does replacement of an arginine for a lysine have an effect on substrate binding to AspAT?(c4)Of the mutant enzymes in the table above, substitution of Arg292 by lysine is always asso-ciated with greatly weakened binding affinity of the substrate and decreased specificity. In the diagramabove, examine the structural relationships between the two carboxylate groups of maleate with Arg-292 and Arg386. Consider the change in binding relationships that maleate must undergo when Arg-292 is mutated into a lysine in the AspAT(Lys292Arg386) enzyme and in the AspAT-(Lys292Lys386)enzyme. On the basis of the diagram above, describe the structural changes that must occur in eachmutant giving rise to weaker substrate binding.

In order for an enzyme to properly bind its substrate and catalyze the reaction, several binding…

Answered: (c2) Identify the active site residues…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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Lysozyme is an antibacterial enzyme found in animals. Residues Asp 101 and Arg 114 are required for efficient catalysis, although they are located at some distance from the active site Glu 35 and Asp 52. Substituting Ala for either Asp 101 or Arg 114 does not significantly alter the enzyme's tertiary structure, but significantly reduces its catalytic activity. Explain.
. The mechanism for lysozyme cleavage of its polysaccharide substrate requires Glu35 in its nonionized form, whereas the nearby Asp52 must be ionized (see the figure below). The pK values for the side-chain carboxyl groups on the two amino acids in solution are virtually identical. a) How can one carboxyl group be charged and the other uncharged in the active site of lysozyme? b) The pH optimum for lysozyme is about 5. Why do you suppose that the activity decreases above and below this optimum? Glu3s NAG Asp52 0-H Glu35 -C Asp52 tri-NAG NAG
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.…
Amino transferases catalyze the transfer of amino groups to alpha-keto acids with pyridoxal phosphate as the prosthetic group. Pyridoxal phosphate can accept an amino group, and its aminated form, pyridoxamine phosphate, can donate its amino group to an alpha-keto acid. Illustrate the detailed mechanism of the transamination reaction in the active site of an aminotransferase. Use the glutamate oxaloacetate transaminase as an example.
What would be the effect on the activity of phosphofructokinase of the mutation of Asp103 to the unusual amino acid shown below? Explain in terms of actual structures of the side chains of Asp and this unusual amino acid.
Amino transferases catalyze the transfer of amino groups to alpha-keto acids with pyridoxal phosphate as the prosthetic group. Pyridoxal phosphate can accept an amino group, and its aminated form, pyridoxamine phosphate, can donate its amino group to an alpha-keto acid. Draw the detailed mechanism of the transamination reaction in the active site of an aminotransferase. Use the glutamate oxaloacetate transaminase as an example.
B-lactamase is an enzyme found in many antibiotic-resistant bacteria that hydrolyzes and inactivates antibiotics like penicillin and cephalosporin. The amount of antibiotic hydrolyzed in 1 minute in a 10-ml solution containing purified ß-lactamase was measured as a function of antibiotic concentration. The kinetics of hydrolysis was performed for two antibiotic substrates (A and B). Assume that the concentration of ß-lactamase was kept constant during the assay. 12 Initial Velocity (nanomole/min) 10 8 2 0 10 20 | 30 [Antibiotic] (µm) 40 50 Antibiotic A Antibiotic B a) Based on the enzyme description, what type of enzyme is ß-lactamase? Lyase Isomerase Ligase Hydrolase Oxidoreductase Transferase b) Based on your answer in (a), what other reactant, in addition to the antibiotic substrate, needs to be in the active site of ß-lactamase for the hydrolysis reaction to proceed? c) From the reaction curves above, what is the approximate value of Vmax for the enzyme reaction? (Do not forget the…
Serine Proteases, such as chymotrypsin, contain Ser/Asp/His as a catalytic triad (i.e. three collaborating amino acids) of amino acids involved in enzyme catalysis. Why is this? Examine each amino acid of this catalytic triad (schematically detailed below: Ser195/Asp102/His57) and, based on your understanding of amino acid characteristics, predict what the role of each amino acid could be. His57 Ser195 Asp102
The enzymatic activity of lysozyme is optimal at pH 5.2 and decreases above and below this pH value. Lysozyme contains two amino acid residues in the active site essential for catalysis: Glu35 and Asp52. The pK value for the carboxyl side chains of these two residues are 5.9 and 4.5 respectively. What is the ionization state of each residue at the pH optimum of lysozyme? How can the ionization states of these 2 amino acid residues explain the pH-activity profile of lysozyme?
#1 Specify the role each of the following amino acids play within the crystal structure and/or active site for Be as specific as possible, with pictures (and mechanistic arrows) as necessary. His11 Arg140 Glu89 Trp68 #2 Provide a step-wise mechanism for the reaction Bisphosphoglycerate mutase catalyzes, using the amino acids responsible for aiding in catalysis. You do not need to add surrounding amino acids that aid in substrate specificity. (drawn out)
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
Using the catalytic mechanism of serine proteases, draw and label a reaction coordinate diagram/graph of the chymotrypsin-catalyzed hydrolysis of a peptide bond.

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