Chapter 6

Protein Function : Enzymes

Part 1

Enzymes

– Physiological significance of enzymes– Catalytic power of enzymes– Chemical mechanisms of catalysis– Mechanism of chymotrypsin– Description of enzyme kinetics and inhibition

Enzyme Learning Goals: to Know

Enzymes

Mostly Proteins (a few RNA’s are capable of catalysis)Active Site: Substrate Binding + Reaction Products

Some require Cofactors (metals) or Coenzymes (organic cpds)

Some enzymes have other binding sites…involved in regulation, we will see later, Part 2

EOC Problem 1 involves the sweetness of corn affected by corn enzymes and Problem 2 calculates the average molar concentration of enzymes in a bacterial cell: you can take it further to find the number of molecules of each enzyme present in a cell.

First Cell Free Prep First to Crystallize Urease Weak bonding at active site results in catalysis

Enzyme Pioneers

Why biocatalysis over inorganic catalysts?• Greater reaction specificity: avoids side products• Milder reaction conditions: conducive to conditions in cells• Higher reaction rates: in a biologically useful timeframe• Capacity for regulation: control of biological pathways

COO

OH

O COO

COO

O COO

NH2

OOCCOO

O

OH

OH

COO

NH2

COO

-

-

-

-

-

-

--

Chorismate mutase

• Metabolites have many potential pathways of decomposition

• Enzymes make the desired one most favorable

EOC Problem 4: Examines the thermal protection of hexokinase that a substrate brings to the table: maintaining conformation under harsh conditions. Later in Part 2 of this chapter we will see X ray data backing this up.

Enzymatic Substrate Selectivity: Phenylalanine hydroxylase

No binding

OOC NH3

H

OOC NH3

H

H NH

HOH

OH

H

OH

CH3

OOC NH3

HOH

--

-

+

+

+

Binding but no reaction

Class Is the First Part of E.C. Number

EC 2.7.1.1 = ATP:glucose phosphotransferase or Hexokinase

2 = Transferase

7 = Phosphotransferase

1 = Transferred to a hydroxyl

1 = Glucose is the acceptor

Enzyme Search By

Class

FMNH2 + O2 + RCHO FMN + RCOOH + H2O + light

Bacterial Luciferase Rxn

Continuing with the EC Numbers-1

Continuing with EC Numbers-2

NiceZyme

Enzyme with an Active Site

Chymotrypsin

Active Site

Thermodynamics of a Reaction

S + E ES E + P

Enzyme Catalyzed Reaction

E + S ↔ ES ↔ EP ↔ E + P

EOC Problem 3: A rate enhancement problem using Urease, the enzyme that converts: Urea CO2 + 2 NH3. The calculation demonstrates how long it would take if urease were not present !

Dihydrofolate ReductaseSubstrate Binds in a Fold or Pocket

ΔGB = binding energy

Folic Acid

NADP+

+

Enzyme Reactions Bind Substrate then Change Shape to Transition State

Triose Phosphate Isomerase

Terribly Slow rate with Glyceraldehyde…phosphate important in stabilizing binding.

Rate Enhancement Due to Proximity (Entropy Reduction)

Acid/Base Catalysis

Catalytic Mechanisms

– acid-base catalysis: give and take protons– covalent catalysis: change reaction paths– metal ion catalysis: use redox cofactors, pKa shifters

– electrostatic catalysis: preferential interactions with Transition State.

Acid Base Catalysis – Involve Proteins R groups

Formation of a Covalent Intermediate

Michaelis Menten Curve

Michaelis Menten Equation:

Vmax [S]

Km + [S]vo =

L. Michaelis and Miss Maud L. Menten. 1913. "Die Kinetik der Invertinwerkung" Biochemische Zeitschrift Vol. 49.

Invertase Reaction: sucrose + H2O glucose + fructose

Michaelis Menten ExperimentMeasure Rate (v) at several concentrations of Substrate (S)

Here is one tube with one beginning concentration of S

Calculate Δ[S]/min or Δ[P]/min.

S PE

This enzyme, triosephosphate isomerase is a one substrate, one product enzyme.

Michaelis Menten Experiment: Real Data

At each [S], the concentration of enzyme is exactly the SAME.

Calculate Δ[S]/min for each [S]

EOC Problem 6 is about using 340nm light to measure dehydrogenase reactions…the classic lactate dehydrogenase.

Do this at more concentrations of S to get a larger data set used for

Initial Velocites are the Dashed Line

A

Michaelis Menten Plot

Michaelis Menten Equation is Non-Linear

Straightened Out by reciprocals…Lineweaver Burke Equation:

1/vo = (KM/Vmax)(1/[S]) + 1/Vmax

the Equation of a Straight Liney = mx + b

Thus, y = 1/vo , x = 1/[S] and m (the slope) = KM/Vmax

Lets Plot this Out…next slide

Vmax [S]

KM + [S]vo =

Lineweaver-Burke PlotDouble Reciprocal

Origin is Zero

Data Points are in this range

There Are Other Equations to Convertthe Michaelis Menten Equation to a

Straight Line

Eadie Hofstie

v = -Km(v/S) + Vmax

Hanes Wolf:

S/v = (1/Vmax)(S) + Km/Vmax

all are y = mx + b

KM = is an Intrinsic Property of an enzyme

What does this mean? Intrinsic vs Extrinsic?

Vmax is an Extrinsic Property of Enzymes

At a high [S], varying only the enzyme conc :

kcat comes from Vmax and [Enz] Vmax is [molar]/sec

[Enz] in molar

To get an Intrinsic Catalytic Constant from Vmax

kcat = Vmax/ [Enz]

kcat/Km

Calculation of Km and VmaxThe enzyme, Practicase

Studentose Productate

Studentose, mM velocity, μmoles/ml/sec

1 12

2 20

4 29

8 35

12 40Assay volume = 1 ml/tube

What’s in the tube: buffer + enzyme, then add substrate at time Zero.

EOC Problems 11(dead easy to do by inspection) and 13 to do by Lineweaver Burke plot

Calculation of Km and Vmax

Studentose, mM 1/[S] Velocity, 1/v

μmoles/ml/sec

1 1 12 0.083

2 0.5 20 0.050

4 0.25 29 0.034

8 0.125 35 0.029

12 0.083 40 0.025

Now Plot this on Lineweaver Burk Plot….remember Zero is near the middle of the graph!

Lineweaver Burke Plot of Practicase

1

1/

Practicase kcat = an Intrinsic Property

In the enzyme assay (one ml), each tube had 10 μg of practicase. The molecular weight of practicase is 20,000 D.

Thus, each tube had

10 μg / 20,000 μg/μmole = 0.0005 μmole practicase

kcat = Vmax/ [Enz] = (50 μmole/sec)/ 0.0005 μmole = 1 x 105 s-1

Thus one enzyme reaction takes 1/ 1x 105 s-1 = 10-5 sec

or 10 μ sec.

What is Wrong with this L-B graph?

What is Wrong with this L-B graph?

Things to Know and Do Before Class

1. Principles of catalysis.

2. Enzyme naming concepts.

3. Intrinsic and Extrinsic values of Enzyme kinetics.

4. Be able to do a Michaelis Menten graph.

5. Be able to do a Lineweaver Burke graph.

6. To do EOC problems 1-6, 11, 13.