Enzymes

US T FOP – DE PARTME NT OF BI OCHE MI S TRY

Enzymes – biological catalysts
Biomolecules (*Proteins) with catalytic activity
Mediate biochemical reactions at efficient rate (vs. inorganic catalysts
eg. Pd and Pt) to sustain life
Substrate- and reaction- specific

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Maximum Catalytic Activity
1. All the active sites of the enzyme is saturated with the substrate.
2. Optimum pH
3. Optimum temperature

1 Km 1 1
= +
𝜈 Vmax [S] Vmax

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Classification of Enzymes

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Invertase
Yeast derived enzyme
Official name: β-fructofuranosidase (EC 3.2.1.26)
Hydrolyses α1β2 glycosidic bond in sucrose
Classified as hydrolase – catalyzing hydrolysis of the terminal
nonreducing β-fructofuranoside residues
Splits sucrose to glucose and fructose
C12H22O11 + H2O  C6H12O6 + C6H12O6
Sucrose glucose fructose

d(+) = + 66.5° d(+) = +52.5° l(-) = -92°

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Enzyme Commission Number

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REACTION MECHANISM

SUCROSE

GLUCOSE

FRUCTOSE

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Dinitrosalicylic Acid Assay
Dinitrosalicyclic acid (DNS) Assay – this method is used to monitor
enzyme activity.

Principle Involved:
DNS (3,5-dinitrosalicylic acid, IUPAC name 2-hydroxy-3,5-
dinitrobenzoic acid) reacts with reducing sugars (eg. glucose and
fructose) to form 3-amino-5-nitrosalicylic acid (ANS). DNS does not
react with sucrose (non-reducing sugar).

In the DNS assay, rate of reaction (enzyme activity) is monitored

(colorimetrically) by measuring the amount of reaction products
(reducing sugars - equimolar mixture of glucose and fructose) that
react with DNS reagent.

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Dinitrosalicylic Acid (DNS) Assay
Reagents for DNS assay
0.5N KOH – to neutralized excess acid
1% DNS reagent
◦ Dinitrosalicylic acid – oxidizing agent
◦ Na2SO3 – stabilizes the red color
◦ NaOH – increases the reactivity of sugars; changes the pH of the
reaction vessel (along with ANS production) halting the invertase
reaction.

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DNS Assay (REDOX)

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Factors Affecting Enzyme Activity
pH
pH dependence of enzyme activity is a consequence of acid-base
behavior or changing degree of ionization of groups in the
enzyme, in the substrate, or in both.
Invertase exhibits high activity over a broad pH range 3.5-5.5 with
optimum pH near 4.5.

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Factors Affecting Enzyme Activity
Temperature
Rate of reaction increases with temp; within the temp range in
which the enzyme is stable and retains its full activity. When temp
range is beyond the optimum, enzymes are denatured followed by
a decrease in enzyme activity.
Optimum temp of invertase = 55oC

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Factors Affecting Enzyme Activity
Enzyme concentration (when [S] is constant)
Substrate concentration (when [E] is constant)
Enzyme inhibitors/activators
Cofactor/Coenzyme

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Dealing with Actual Data
Standard Sugar Solutions

Test Tube 0 1 2 3 4 5 6
Invert Sugar (0.01 M, mL) 0.00 0.10 0.30 0.50 0.80 1.00 1.20
Distilled Water (mL) 2.50 2.40 2.20 2.00 1.70 1.50 1.30
acetate buffer (pH 4.70), mL 0.50 0.50 0.50 0.50 0.50 0.50 0.50
Total Volume/test tube 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Total Volume
Concentration of Invert Sugar, mM 0 0.33 1.00 1.67 2.67 3.33 4.00 y
Amount of Invert Sugar, mmol 0 1.00 3.00 5.00 8.00 10.00 12.00 y
Concentration of Invert Sugar Standard, M 0.01
Absorbance (540 nm) 0.167 0.278 0.315 0.378 0.478 0.596 0.688
Absorbance (540 nm) -
0 0.111 0.148 0.211 0.311 0.429 0.521
Corrected x

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 Standard Curve
Concentration of Invert Sugar, mM 0 0.33 1.00 1.67 2.67 3.33 4.00 y
Amount of Invert Sugar, mmol 0 1.00 3.00 5.00 8.00 10.00 12.00 y
Concentration of Invert Sugar Standard, M 0.01
Absorbance (540 nm) 0.167 0.278 0.315 0.378 0.478 0.596 0.688
Absorbance (540 nm) -
0 0.111 0.148 0.211 0.311 0.429 0.521
Corrected x

Invert Sugar Standard Curve y = 0.1136x + 0.0424 y = 0.0379x + 0.0424

R² = 0.9752 Invert Sugar Standard Curve R² = 0.9752
0.6 0.6
Absorbance (540 nm)

Absorbance (540 nm)

0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0.00 1.00 2.00 3.00 4.00 5.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Invert Sugar Concentration, mM Invert Sugar Amount, mmol

Plotting Absorbance as a function of concentration (or amount ~ mmol) will

essentially give the similar equation (except for the slope) of the line.

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Effect of pH y = 0.1136x + 0.0424
R² = 0.9752

Effect of pH
pH 2 3 5 7 8 12 y
Absorbance (intact enzyme) 0.267 0.357 0.546 0.425 0.368 0.225
Absorbance (denatured enzyme) 0.137 0.115 0.129 0.128 0.125 0.132
Absorbance (corrected) 0.267 0.357 0.546 0.425 0.368 0.225 x

Buffer Enzyme Soln Substrate distilled H2O Total

Volume per test tube (mL) 0.50 0.10 1.00 1.40 3.00

Concentration (mM) 1.98 2.77 4.43 3.37 2.87 1.61 x

Amount (mmol) 5.93 8.31 13.30 10.10 8.60 4.82 x

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Effect of Temp y = 0.1136x + 0.0424
R² = 0.9752

Effect of Temperature
Temp 25 30 50 60 70 90 y
Absorbance 0.249 0.295 0.455 0.528 0.428 0.207
Absorbance (denatured
0.125 0.125 0.125 0.125 0.125 0.125
enzyme)
Absorbance (corrected) 0.124 0.17 0.33 0.403 0.303 0.082

Enzyme
Buffer Substrate d. H2O Total
Soln
Volume per test tube (mL) 0.50 0.10 1.00 1.40 3.00

Concentration (mM) 0.72 1.12 2.53 3.17 2.29 0.35 x

Amount (mmol) 2.15 3.37 7.60 9.52 6.88 1.05 x

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End.

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