We want to measure the activity of alanine aminotransferase (ALAT) present in aserum. The reaction catalyzed by the enzyme is:Reaction 1:+H3N-glutamateHCCH₂CH₂COO-COO +pyruvateCH3C=00.1 M phosphate buffer pH 7.4 : 550 µL1.2 M alanine : 100 μLCH3time (min)A340COOCOO™pyruvatelactate dehydrogenase* (LDH, 300 µg.mL-¹): 50 μL1.5 mM NADH: 200 μL0.04 M a-ketoglutarate: 500 μLserum containing ALAT: 600 μLThe enzyme reaction is realized in the following conditions:In a 1 cm-cuvette are added:00.915ALATNADH + H+LDHa-cétoglutarateCOO* Lactate dehydrogenase (LDH) reduces pyruvate into lactate, with the concomitantoxydation of NADH. This allows to indirectly measure the amount of product formed.Reaction 2:NAD+10.741C=OHCH₂CH₂COO™CH3C-OHCOOlactateThe reaction is performed at 25 °C and the absorbance at 340 nm is monitored everyminute, for 5 min. The absorbance values are given in the table below:Data:alanineENADH at 340 nm = 6220 M¹.cm1.One international unit corresponds to the amount of enzyme that catalyzes theconversion of 1 µmole of substrate to product in one minute.*H3N C COO320.572 0.402CH340.23350.1591- In which sense does the reaction take place?2- Calculate alanine, LDH, NADH and a-ketoglutarate concentrations in the reactionmedium at t = 0 (in the same units as the initial concentrations).3- Draw the graph A340 = f(t). Comment the shape of the curve, particularly theportion that corresponds to a plateau.

There are two reactions involved in the situation:Reversible reaction: glutamate + pyruvate…

Answered: In which sense does the reaction take…

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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To determine whether this organism is capable of performing carbohydrate metabolism, ateam of researchers of which you are a part wanted to isolate an enzyme calledglyceraldehyde 3-phosphate dehydrogenase (GAPDH). The enzyme has the followingproperties. Number of amino acids: 335 Molecular Weight: 36,053 DapI: 8.57 Your project leader wanted you to present a basic purification protocol of GAPDH from theorganism based on the given properties above. Give a 1-sentence explanation for each stepof your protocol.
Chitinase is a protein that breaks down chitin, a primary component of the cell wall in fungi, scales in fish and exoskeletons of arthropods. The activity of chitinase extracted from a plant was shown to be optimum at pH 5. You were tasked to prepare 300 mL of 150 mM buffer solution for further analysis of the extracted chitinase. REAGENTS Ka 2.5M Acetic acid Solid NaOAc•3H2O [136.08g/mol] 1.76 x 10-5 2.5M NH3 Solid NH4Cl [53.49g/mol] 5.6 x 10-10 2.5M Lactic acid Solid sodium lactate [112.06g/mol] 4.0 x 10-5 5 M HCl 5M NaOH Pls show sol'ns 1. Given the following reagents, give the moles of each component (acid & base).2. What are the mass/volume of the components needed to prepare the buffer? 3. What will the pH of the buffer be if 1mL of 5 M NaOH was added?
The KM for the reaction of chymotrypsin with N-acetylvaline ethyl ester is 8.8 × 10−2 M, and the KM for the reaction of chymotrypsin with N-acetyltyrosine ethyl ester is 6.6 × 10−4 M. (a) Which substrate has the higher apparent affi nity for the enzyme? (b) Which substrate is likely to give a higher value for Vmax?
The turnover number of the enzyme fumarase that catalyzes the reaction, Fumarate + H20 ===→ L-malate, is 2.5 x 103 S - l and Km = 4.0 X 10- 6 mol/L. Calculate the rate of conversion of fumarate to L-malate if the fumarase concentration is 1.0 x 1 0 - 6 mol/L and the fumarate concentration is 2.04 x 10- 4 mol/L.
Shown below is a proposed mechanism for the cleavage of sialic acid by the viral enzyme neuraminidase. The kcat for the wild-type enzyme at pH =6.15, 37 °C is 26.8 s-1.(a) Describe the roles of the following amino acids in the catalytic mechanism: Glu117, Tyr409, and Asp149. List all of the following that apply:general acid/base catalysis (GABC), covalent catalysis, electrostaticstabilization of transition state.(b) Based on the information shown in the scheme, would you expect mutation of Glu 117 to Ala to have a greater effect on KM or kcat?(c) For the R374N mutant at pH = 6.15, 37 °C, kcat is 0.020 s-1, and KMis relatively unaffected. Based on this result, it seems that R374 is morecritical for catalysis than for substrate binding. Explain how R374 stabilizesthe reaction transition state more than the substrate (i.e., what feature of this reaction would explain tighter binding to the transition state vs. substrate?).
In the first step of the aldolase reaction, an active site Lys229 residue, with its side chain amino group in the deprotonated state, acts as a nucleophile and attacks the carbonyl C2 carbon of fructose 1,6-bisphosphate to form a Schiff base (boxed in the scheme). Since the pKa of the Lys side chain amino group in free solution is ~10.5, the pKa of Lys229 side chain must have been perturbed to a (higher lower) value for the enzyme to be active at neutral pH. the answer should include sufficient details, including the definition of pKa.
The α-amylase (α-AM) enzyme secreted by Aspergillus has an isoelectric point of approximately 4.2. If the purification method in problem 1 operates at pH 6.5, would the protein carry a positive charge, negative charge, or net zero charge?
Phosphatidylserine (PS) is considered to be an intermediate in the biosynthesis of phosphatidylethanolamine (PE) in E. coli, yet PS is not found in appreciable amounts among E. coli membrane phospholipids. Because PS must be present in the membrane to serve as an intermediate, how might you explain its failure to accumulate to a significant extent? What kinds of experiments could test your proposed explanation?
The active site of an enzyme that uses a general acid-base catalytic mechanism contains a Glu and an Asp residue (both of which are essential for catalysis) with pKa values of 5.9 and 4.5, respectively. If the enzyme is found in the lysosome (pH = 5.2), which residue will act as the general acid and which will act as the general base during the initial steps of the reaction?
Consider the binding reaction L + R → LR, where L is a ligand and R is its receptor. When 1 × 10−3 M of L is added to a solution containing 5 × 10−2 M of R, 90 percent of the L binds to form LR. What is the Keq of this reaction? How will the Keq be affected by the addition of a protein that facilitates (catalyzes) this binding reaction? What is the dissociation equilibrium constant Kd?
complete the following mechanism showing the cleavge of the peptide bond for the following cysteine protease reaction. you only need a catalytic dyad for cystein proteases because the pKa of cysteine is low enough (8) and close enough to the pH of 7.4 that is can easily be deprotonated by histidine.
The Keq (25C) of the reaction below is 635.67. Fructose 1,6-biphosphate <-->fructose -6-phosphate + Pi. a) What is the standard Gibbs free energy change for this reaction? b) if the concentrationof fructose 1,6 biphosphate is adjusted to 0.85 M and that of fructose 6 phosphate and phosphate adjusted to 0.055 M, what is the actual free energy change

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