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    Universidad Del Atlántico Chemical Engineering Program Bioprocess Course 2022 Homework of Enzymatic Kinetic

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    CAMILA ANDREA PEREZ VELASQUEZ
    An enzyme that converts a single substrate to a single product was isolated and its properties were analyzed. The molecular mass was determined to be approximately 23 kDa based on SDS gel electrophoresis. Absorbance and amino acid analysis were used to calculate the enzyme concentration and minimum molecular mass. Kinetic experiments were performed with varying substrate concentrations in the presence and absence of inhibitors to determine kinetic parameters such as Km, Vmax, and inhibition constants.

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    Universidad Del Atlántico Chemical Engineering Program Bioprocess Course 2022 Homework of Enzymatic Kinetic

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    CAMILA ANDREA PEREZ VELASQUEZ
    61 views3 pages
    An enzyme that converts a single substrate to a single product was isolated and its properties were analyzed. The molecular mass was determined to be approximately 23 kDa based on SDS gel electrophoresis. Absorbance and amino acid analysis were used to calculate the enzyme concentration and minimum molecular mass. Kinetic experiments were performed with varying substrate concentrations in the presence and absence of inhibitors to determine kinetic parameters such as Km, Vmax, and inhibition constants.

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    An enzyme that converts a single substrate to a single product was isolated and its properties were analyzed. The molecular mass was determined to be approximately 23 kDa based on SDS gel electrophoresis. Absorbance and amino acid analysis were used to calculate the enzyme concentration and minimum molecular mass. Kinetic experiments were performed with varying substrate concentrations in the presence and absence of inhibitors to determine kinetic parameters such as Km, Vmax, and inhibition constants.

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    61 views3 pages

    Universidad Del Atlántico Chemical Engineering Program Bioprocess Course 2022 Homework of Enzymatic Kinetic

    Uploaded by

    CAMILA ANDREA PEREZ VELASQUEZ
    An enzyme that converts a single substrate to a single product was isolated and its properties were analyzed. The molecular mass was determined to be approximately 23 kDa based on SDS gel electrophoresis. Absorbance and amino acid analysis were used to calculate the enzyme concentration and minimum molecular mass. Kinetic experiments were performed with varying substrate concentrations in the presence and absence of inhibitors to determine kinetic parameters such as Km, Vmax, and inhibition constants.

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    Prof. José A.

    Pérez

    Universidad del Atlántico


    Chemical Engineering Program
    Bioprocess course 2022

    Homework of enzymatic kinetic

    A new enzyme (E) has been isolated and purified which converts a single
    substrate (S) into a single product (P). A Chemical Engineer Student determined
    Mr (molar mass) by gel filtration as ~ 46,400. However, in SDS gel electrophoresis,
    a molecular mass of ~ 23 kDa was indicated for the single protein band observed.
    A solution of the enzyme was analyzed in the following way. The absorbance at
    280 nm was found to be 0.512. A 1.00 ml portion of the same solution was
    subjected to amino acid analysis and was found to contain 71.3 nmol of
    tryptophan. N-terminal analysis on the same volume of enzyme revealed 23.8
    nmol of N-terminal alanine. The following information can be useful to
    calculate the enzyme concentration and minimum molar mass taking
    account into each amino acid by separated.

    [ E]A =280nm=1.55 A1280


    cm
    nm [mg/ml]
    m Mr AA
    Mr minimum= E
    m AA
    Where :
    mE =Enzyme mass ; mAA = Amino acid mass and Mr AA =Molar mass of amino acid

    1. What is the approximate molecular mass of the enzyme? Discuss this


    answer. Be sure to use an appropriate number of significant figures in this
    and other calculations.
    2. What is the concentration of your enzyme in moles per liter of active sites?
    3. What is the molar extinction coefficient ε at 280 nm where A = εcl; A =
    absorbance, c = mol / liter, and l = cell width in cm. Assume that all
    spectrophotometric measurements are made in 1.00 cm cuvettes.

    A second preparation of the enzyme had an absorbance at 280 nm of 0.485. This


    enzyme was diluted very carefully: 1.00 ml into 250 ml and this diluted enzyme
    was used for the following experiments (I to III).

    Experiment I. A 1.00 ml portion of the diluted enzyme was added to 250 ml of


    buffered substrate at pH 7.0 and was mixed rapidly. The resulting initial substrate
    concentration [S] o was 1.000 mM. This reaction mixture was held at 25.0°C and
    portions were removed periodically at time t for analysis of the product P formed.
    The results follow. Plot [P] vs. time.

    What is km, vm and kcat?


    Prof. José A. Pérez

    Experiment II. In a second experiment, a series of test tubes were set up, each
    containing a different amount of buffered substrate at pH 7 but each in a volume of
    exactly 4.00 ml. The same enzyme solution used above (absorbance at 280 nm =
    0.485) was diluted 2.00 ml in 250 ml as in I, then again 2.00 ml in 200 ml. Portions
    of 1.00 ml of this twice diluted enzyme were added at t = 0 to each of the test tubes
    of buffered substrate. The reaction was stopped in just 10.0 minutes by adding
    perchloric acid; a suitable reagent was added to provide for a colorimetric
    determination of the product. The results were as follows:

    1. Plot 1/v vs 1/ [S] where v is in units of µmol per tube and [S] in
    millimoles/liter. Evaluate km, vm , and kcat from this plot.
    2. Plot the same data as v/[S] vs. v. Again evaluate k m and vm.

    Experiment III. The preceding experiment was repeated but an inhibitor was
    present in each tube in a concentration equal to 5.00 mM, 10.0 mM, or 25.0 mM.
    Two different inhibitors were used, A and B. The following results were obtained.

    1. Plot l/v vs 1/[S] for each of these sets of data. For each case evaluate v m,
    apparent km, and inhibitor constants KI.
    2. For uninhibited enzymes, when [S] = 0.4 mM what fraction of the enzyme is
    ES? Free E?
    Prof. José A. Pérez

    3. For enzyme in the presence of 10.00 mM inhibitor and 0.8 mM substrate,


    what fraction of the total enzyme is ES? EI? free E?

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