KAPLAN_USMLE_STEP_1_LECTURE_NOTES_2018_BIOCHEMISTRY_and_GENETICS

Part I ● Biochemistry

Note

Hydrolysis of high-energy bonds in ATP or GTP provide energy to drive reactions in which ∆G >0.

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BIOCHEMICAL REACTIONS

Chemical reactions have 2 independent properties: energy and rate.

∆G represents the amount of energy released or required per mole of reactant. The amount or sign of ∆G indicates nothing about the rate of the reaction.

Table I-8-2. Energy versus Rate

	Energy (∆G)			Rate (v)
Not affected by enzymes			Increased by enzymes
∆G <0, thermodynamically spontaneous			Decrease energy of activation, ∆G‡
(energy released, often irreversible)
∆G >0, thermodynamically
nonspontaneous (energy required)
∆G = 0, reaction at equilibrium (freely
reversible)
∆G0 = energy involved under
standardized conditions

The rate of the reaction is determined by the energy of activation (∆G‡), which is the energy required to initiate the reaction. ∆G and ∆G‡ are represented in the figure below. Enzymes lower the energy of activation for a reaction; they do not affect the value of ∆G or the equilibrium constant for the reaction, Keq.

Enzyme catalyzed

Uncatalyzed

Energy	Gs						G‡
Free
						G
	Gp

Reaction Progress

Figure I-8-4. Energy Profile for a Catalyzed and Uncatalyzed Reaction

Michaelis-Menten Equation

The Michaelis-Menten equation describes how the rate of the reaction, V, depends on the concentration of both the enzyme [E] and the substrate [S], which forms product [P].

E + S E - S → E + P
V =	k2[E ] [S]	or, with [E] held constant, V =	Vmax [S]
			Km + [S]
	Km + [S]
Note: Vmax = k2[E]

Chapter 8 ● Amino Acids, Proteins, and Enzymes

Vmax is the maximum rate possible to achieve with a given amount of enzyme.

The only way to increase Vmax is by increasing the [E]. In the cell, this can be accomplished by inducing the expression of the gene encoding the enzyme.

The other constant in the equation, Km is often used to compare enzymes. Km is the substrate concentration required to produce half the maximum velocity. Under certain conditions, Km is a measure of the affinity of the enzyme for its substrate. When comparing two enzymes, the one with the higher Km has a lower affinity for its substrate. The Km value is an intrinsic property of the en- zyme-substrate system and cannot be altered by changing [S] or [E].

When the relationship between [S] and V is determined in the presence of constant enzyme, many enzymes yield the graph shown below, a hyperbola.

	50		Vmax
mol/sec)(
	25
V

Km

2

4

6

8

10

[S] (mM)

Figure I-8-5. Michaelis-Menten Plot

Recall Question

Which of the following conditions will result in arginine becoming an essential amino acid?

A.Diabetes

B.Pregnancy

C.Sepsis

D.Starvation

Answer: B

Bridge to Medical Genetics

A missense mutation in the coding region of a gene may yield an enzyme with a different Km.

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Part I ● Biochemistry

Lineweaver-Burk Equation

The Lineweaver-Burk equation is a reciprocal form of the Michaelis-Menten equation. The same data graphed yield a straight line, as shown below.

The actual data are represented by the portion of the graph to the right of the y-axis, but the line is extrapolated into the left quadrant to determine its intercept with the x-axis. The intercept of the line with the x-axis gives the value of –1/Km. The intercept of the line with the y-axis gives the value of 1/Vmax.

1 v (sec/ mol)

	1	=		Km		1		+		1
	V		V		[S]				V
				max						max

0.06

0.04

0.02	1
	Vmax

Note

Many drugs are competitive inhibitors of key enzymes in pathways.

•The statin drugs (lovastatin, simvastatin), used to control blood cholesterol, competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in cholesterol biosynthesis.

•Methotrexate, an antineoplastic drug, competitively inhibits dihydrofolate reductase, depriving the cell of active folate needed for purine and deoxythymidine synthesis, thus interfering with DNA replication during S phase.

An example of a noncompetitive inhibitor is allopurinol, which noncompetitively inhibits xanthine oxidase.

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					0
:1.0		:0.5				0.5			1.0
:		1						1		(mM:1)
		Km						[S]

Figure I-8-6. Lineweaver-Burk Plot

Inhibitors and Activators

Competitive inhibitors resemble the substrate and compete for binding to the active site of the enzyme. Noncompetitive inhibitors do not bind at the active site; they bind to regulatory sites on the enzyme.

Table I-8-3. Important Classes of Enzyme Inhibitors

	Class of Inhibitor			Km			Vmax
	Competitive		Increase				No effect
	Noncompetitive		No effect				Decrease

The effects of these classes of inhibitors on Lineweaver-Burk kinetics are shown below. Notice that on a Lineweaver-Burk graph, inhibitors always lie above the control on the right side of the y-axis.

1
V	; Inhibitor
	No inhibitor present
0	1
	[S]

Figure I-8-7. Lineweaver-Burk Plot

of Competitive Inhibition

Chapter 8 ● Amino Acids, Proteins, and Enzymes

1	; Inhibitor
V
	No inhibitor present
0	1
	[S]

Figure I-8-8. Lineweaver-Burk Plot

of Noncompetitive Inhibition

Figure I-8-9 shows the effect on a Lineweaver-Burk plot of adding more enzyme. It might also represent adding an activator to the existing enzyme or a covalent modification of the enzyme. An enzyme activator is a molecule that binds to an enzyme and increases its activity. In these latter two cases the Km might decrease and/or the Vmax might increase but the curve would always be below the control curve in the right-hand quadrant of the graph.

1		Enzyme + substrate
		control curve
	V

Add more enzyme, or activator

		1
0
		[S]

Figure I-8-9. Lineweaver-Burk Plot Showing the Addition of More Enzyme or the Addition of an Activator

Cooperative Enzyme Kinetics	High-Yield

Certain enzymes do not show the normal hyperbola when graphed on a Michaelis-Menten plot ([S] versus V), but rather show sigmoid kinetics owing to cooperativity among substrate binding sites. Cooperative enzymes have multiple subunits and multiple active sites. Enzymes showing cooperative kinetics are often regulatory enzymes in pathways (for example, phosphofructokinase-1 [PFK-1] in glycolysis).

In addition to their active sites, these enzymes often have multiple sites for a variety of activators and inhibitors (e.g., AMP, ATP, citrate, fructose-2,6-bispho- sphate [F2,6-BP]). Cooperative enzymes are sometimes referred to as allosteric enzymes because of the shape changes that are induced or stabilized by binding substrates, inhibitors, and activators.

Bridge to Pharmacology

Methanol poisoning (wood alcohol poisoning) is treated with ethanol administration. Both are substrates for alcohol dehydrogenase (ADH), with ethanol having a much lower Km for the enzyme compared with methanol. This prevents methanol from being converted to formaldehyde, which is toxic and not metabolized further.

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Part I ● Biochemistry

Activator

V

Control

Inhibitor

[S]

Figure I-8-10. Cooperative Kinetics

Transport Kinetics

High-Yield

The Km and Vmax parameters that apply to enzymes are also applicable to transporters in membranes. The kinetics of transport can be derived from the Michaelis-Menten and Lineweaver-Burk equations, where Km refers to the solute concentration at which the transporter is functioning at half its maximum activity. The importance of Km values for membrane transporters is exemplified with the variety of glucose transporters (GLUT) and their respective physiologic roles (see Chapter 12).

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Chapter 8 ● Amino Acids, Proteins, and Enzymes

Review Questions

Select the ONE best answer.

1.The peptide ala-arg-his-gly-glu is treated with peptidases to release all of the amino acids. The solution is adjusted to pH 7, and electrophoresis is performed. In the electrophoretogram depicted below, the amino acid indicated by the arrow is most likely to be

–

+

A.glycine

B.arginine

C.glutamate

D.histidine

E.alanine

2.The reaction catalyzed by hepatic phosphofructokinase-1 has a ∆G0 value of –3.5 kcal/mol. This value indicates that under standard conditions this reaction

A.is reversible

B.occurs very slowly

C.produces an activator of pyruvate kinase

D.is inhibited by ATP

E.has a low energy of activation

F.will decrease in activity as the pH decreases

G.cannot be used for gluconeogenesis

H.shows cooperative substrate binding

I.is indirectly inhibited by glucagon

J.is stimulated by fructose 2,6-bisphosphate

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Part I ● Biochemistry

3.The activity of an enzyme is measured at several different substrate concentrations, and the data are shown in the table below.

[S] (mM)	V0 (mmol/sec)
0.010	2.0
0.050	9.1
0.100	17
0.500	50

1.0067

5.0091

10.0	95
50.0	99

100.0100

Km for this enzyme is approximately

A.50.0

B.10.0

C.5.0

D.1.0

E.0.5

4.Which of the diagrams illustrated below best represents the effect of ATP on hepatic phosphofructokinase-1 (PFK-1)?

	+ATP
velocity		velocity		velocity
			+ATP
A		B		C
	Fructose 6-P		Fructose 6-P

+ATP

Fructose 6-P

velocity D

+ATP

Fructose 6-P

velocity E

+ATP

Fructose 6-P

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Chapter 8 ● Amino Acids, Proteins, and Enzymes

5.Several complexes in the mitochondrial electron transport chain contain non-heme iron. The iron in these complexes is bound tightly to the thiol group of which amino acid?

A.Glutamine

B.Methionine

C.Cysteine

D.Tyrosine

E.Serine

Items 6–8

Consider a reaction that can be catalyzed by one of two enzymes, A and B, with the following kinetics.

	Km (M)	Vmax (mmol/min)
A.	5 × 10−6	20
B.	5 × 10−4	30

6.At a concentration of 5 × 10−6 M substrate, the velocity of the reaction catalyzed by enzyme A will be

A.10

B.15

C.20

D.25

E.30

7.At a concentration of 5 × 10−4 M substrate, the velocity of the reaction catalyzed by enzyme B will be

A.10

B.15

C.20

D.25

E.30

8.At a concentration of 5 × 10−4 M substrate, the velocity of the reaction catalyzed by enzyme A will be

A.10

B.15

C.20

D.25

E.30

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Part I ● Biochemistry

9.A worldwide pandemic of influenza caused by human-adapted strains of avian influenza or bird flu is a serious health concern. One drug for treatment of influenza, Tamiflu (oseltamivir), is an inhibitor of the influenza viral neuraminidase required for release of the mature virus particle from the cell surface. Recent reports have raised concerns regarding viral resistance of Tamiflu compelling the search for alternative inhibitors. Another drug, Relenza (zanamavir), is already FDA approved for use in a prophylactic nasal spray form. The graph below show kinetic data obtained for viral neuraminidase activity (measured as the release of sialic acid from a model substrate) as a function of substrate concentration in the presence and absence of Relenza and Tamiflu.

+ Relenza

4

+ Tamiflu

1/V 3

( moles/min)

No inhibitor

2

1

0

10

20

30

40

1/[S] ( M)

Based on the kinetic data, which of the following statements is correct?

A.Both drugs are competitive inhibitors of the viral neuraminidase.

B.Both drugs are noncompetitive inhibitors of the viral neuraminidase.

C.Tamiflu increases the Km value for the substrate compared to Relenza

D.Relenza increases the Vmax value for the substrate compared to Tamiflu.

E.Relenza is not an inhibitor of neuraminidase, but inhibits another viral enzyme.

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Chapter 8 ● Amino Acids, Proteins, and Enzymes

Answers

1.Answer: B. Arginine is the most basic of the amino acids (pI~11) and would have the largest positive charge at pH 7.

2.Answer: G. The negative ∆G0 value indicates the reaction is thermodynamically favorable (irreversible), requiring a different bypass reaction for conversion of F1, 6BP to F6P in the gluconeogenic pathway.

3.Answer: E. Because the apparent Vmax is near 100 mmol/sec, Vmax/2 equals 50 mmol/sec. The substrate concentration giving this rate is 0.50 mM.

4.Answer: B. Sigmoidal control curve with ATP inhibiting and shifting curve to the right is needed.

5.Answer: C. Cysteine has a sulfhydryl group in its side chain. Although methionine has a sulfur in its side chain, a methyl group is attached to it.

6.Answer: A. At the concentration of 5 × 10–6 M, enzyme A is working at

one-half of its Vmax because the concentration is equal to the Km for the substrate. Therefore, one-half of 20 mmol/min is 10 mmol/min.

7.Answer: B. At the concentration of 5 × 10–4 M, enzyme B is working at

one-half of its Vmax because the concentration is equal to the Km for the substrate. Therefore, one-half of 30 mmol/min is 15 mmol/min.

8.Answer: C. At the concentration of 5 × 10–4 M, 100 × the substrate concentration at Km, enzyme A is working at its Vmax, which is 20 mmol/min.

9.Answer: B. Based on the graph, when the substrate is present, Tamiflu

results in the same Vmax and higher Km compared to the line when no inhibitor added. These are hallmarks of competitive inhibitors of enzymes, which Tamiflu is. Noncompetitive inhibitors result in decreased

Vmax and the same Km with no inhibitor added, which is shown by the Relenza line in the graph.

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