BIOCHEM 463 – Relationship to the Blood Buffering System

13 Jun BIOCHEM 463 – Relationship to the Blood Buffering System

Question
Biochemistry for Physiology (BCHM 463),
Fall 2015
This Assignment is worth 50 points and is due on November 2, 2015 (11/2/2015). You may work on the
assignment in groups if desired; however, each student must turn in their own assignment and the
answers must be in their own words.
The following pages comprise several case studies covering some of the topics that have been (or will be)
covered in the BCHM 463 course. For the assignment, please choose from the following:
Amino Acids, Protein Structure and Function, and pH regulation (25 points)
Case 1 or Case 3
Protein Function, Enzymes, and Kinetics (25 points)
Case 13 or Case 20

Case 1
Acute Aspirin Overdose:
Relationship to the Blood Buffering System
Focus concept
The response of the carbonic acid/bicarbonate buffering system to an overdose of aspirin is examined.

Prerequisites
C
C

Principles of acids and bases, including pKa and the Henderson-Hasselbalch equation.
The carbonic acid/bicarbonate blood buffering system.

Background
You are an emergency room physician and you have just admitted a patient, a 23-year-old female,
who had been hospitalized for psychiatric treatment for the past six months. She was out on a day pass
when she was brought to the emergency room around 9 pm. The patient was disoriented, had trouble
speaking, and was suffering from nausea and vomiting. She was also hyperventilating. The patient
admitted to taking an entire bottle of aspirin, which contained 250 tablets. The patient admitted that she
took the tablets around 7 pm that evening. You draw blood from the patient and the laboratory performs
the analyses shown in Table 1.1. The patient is experiencing mild respiratory alkylosis.
Table 1.1: Arterial blood gas concentration in patient
Patient, two hours after
aspirin ingestion

Patient, ten hours after
aspirin ingestion

Normal values

pCO2

26 mm Hg

19 mm Hg

35-45 mm Hg

HCO3-

18 mM

21 mM

22-26 mM

pO2

113 mm Hg

143 mm Hg

75-100 mm Hg

pH

7.44

7.55

7.35-7.45

Blood salicylate concentration, mg/dL

57

117

In the emergency room, the patient is given a stomach lavage with saline and two doses of activated
charcoal to adsorb the aspirin. Eight hours later, nausea and vomiting became severe, and her respiratory
rate increased; she was in severe respiratory alkylosis, and further treatment was required. You carry out
a gastric lavage at pH = 8.5 and administer further activated charcoal treatments, one every 30 minutes.
A bicarbonate drip was required to prevent the blood bicarbonate concentration from dropping below 15
mM. Over the next four hours, blood salicylate concentrations begin to decrease. The patient’s blood
pH begins to drop around 24 hours after the aspirin ingestion and finally returns to normal at 60 hours
after the ingestion.
1

CASE 1 C Acute Aspirin Overdose: Relationship to the Blood Buffering System.

Questions
1. Aspirin, or acetylsalicylic acid (structure shown in Figure 1.1), is hydrolyzed in the presence of aqueous acid and stomach esterases (which act as
catalysts) to salicylic acid (the pharmacologically active form of the drug)
and acetic acid. Write the balanced chemical reaction for this transformation.
2. Since the patient was brought into the emergency room only two hours
after the overdose, you suspect that her stomach might contain undissolved aspirin that is continuing to be absorbed. The fact that she is
experiencing severe respiratory alkylosis 10 hours after the ingestion
confirms your suspicion and you decide to use a gastric lavage at pH 8.5 Figure 1.1: Structure
to effectively remove any undissolved aspirin. This treatment solubilizes of aspirin.
the aspirin so that it can easily be removed from the stomach.
a. Calculate the percentage of protonated and unprotonated forms of salicylic acid at the pH of the
stomach, which is usually around 2.0.
b. Calculate the percentage of protonated and unprotonated forms of salicylic acid at the pH of the
gastric lavage. Why does the gastric lavage result in increased solubility of the drug? (Note:
Assume that the pKa values for the carboxylate group in salicylic acid and acetylsalicylic acid are
the same.)
3. It has been shown that salicylates act directly on the nervous system to stimulate respiration. Thus,
our patient is hyperventilating due to her salicylate overdose.
a. Explain how the salicylate-induced hyperventilation leads to the values of pO2 and pCO2
symptoms seen in the patient.
b. Explain how the salicylate-induced hyperventilation causes the pH of the patient’s blood to
increase. Illustrate your answer with the appropriate equations.
c. Why was the bicarbonate drip necessary?
4. a. Use the Henderson-Hasselbalch equation to determine the ratio of HCO3- to H2CO3 in the
patient’s blood 10 hours after aspirin ingestion. How does this compare to the ratio of HCO3- to
H2CO3 in normal blood? Can the H2CO3/HCO3- system serve as an effective buffer in this patient?
Explain.
b. Compare the concentration of HCO3- in a normal person and in our patient. Then calculate the
concentration of H2CO3 in the patient’s blood 10 hours after aspirin ingestion. Again, compare
this value to the concentration of H2CO3 found normally, and again address the question of buffer
effectiveness in the patient.
5. Sixty hours after aspirin ingestion, the patient’s blood pH has returned to normal (pH = 7.4). Describe
how the carbonic/bicarbonate buffering system responded to bring the patient’s blood pH back to
normal.
2

CASE 1 C Acute Aspirin Overdose: Relationship to the Blood Buffering System.

6. Are there other substances in the blood that can serve as buffers?

Reference
Krause, D. S., Wolf, B. A., and Shaw, L. M. (1992) Therapeutic Drug Monitoring 14, pp. 441-451.

3

Case 3
Carbonic Anhydrase II Deficiency
Focus concept
The role of the carbonic anhydrase enzyme in normal bone tissue formation is examined.

Prerequisites
C
C
C
C

Amino acid structure.
The carbonic acid/bicarbonate blood buffering system.
Membrane transport proteins.
Basic genetics.

Background
In this case, we will consider our patients: three sisters, aged 21, 24, and 29 years of age who are
short of stature and obese. (There is a fourth sister in the family who appears to be normal, as she is taller
than the other three sisters. The parents also appear to be normal.) As children, the symptoms of the three
sisters were similar–delayed mental and physical development, muscle weakness, and renal tubular
acidosis. They frequently suffered bone fractures as children. X-rays showed cerebral calcification and
other skeletal abnormalities. After reviewing the sisters’ medical histories, you draw samples of blood and
send it to the laboratory for analysis. The laboratory reports to you that your patients all have a carbonic
anhydrase II deficiency.
There are seven isozymes of carbonic anhydrase (CA), three of which occur in humans and are
designated CA I, II and III. They are all monomeric zinc metalloenzymes and have molecular weights of
29 kilodaltons. X-ray crystallographic data shows that the enzyme is roughly spherical with the active site
located in a conical cleft. One side of this cleft is lined with hydrophobic amino acid residues while the
other side is lined with hydrophilic residues. The zinc ion is located at the bottom of the cleft and is
coordinately covalently bound to the imidazole rings of three histidine residues.
The carbonic anhydrase II isozyme is found in bone, kidney, and brain, which is why the defects
occur in these tissues when the enzyme is deficient or non-functional. The carbonic anhydrase II enzyme
is highly active, with one of the highest turnover rates of any known enzyme, and is critical in maintaining
proper acid-base balance.

1

CASE 3 C Carbonic Anhydrase II Deficiency

Questions
1. Carbonic anhydrase catalyzes the reaction between water and carbon dioxide to yield carbonic acid.
The carbonic acid then undergoes dissociation. Write the two equations that describe these processes.
What products form when carbonic acid is dissociated?
2. Each of the three sisters with the symptoms described above showed a carbonic anhydrase II
deficiency. In contrast, the fourth sister and both parents showed half-normal levels of the enzyme.
Construct a chart which describes how the carbonic anhydrase deficiency syndrome is inherited. Note
that the defective carbonic anhydrase gene is inherited as an autosomal recessive gene.
3. A genetic analysis of one of the sister’s genes indicates that a (His 6 Tyr) mutation at amino acid 107
is responsible for the carbonic anhydrase deficiency. Using what you know about amino acid
structure, propose a hypothesis that might explain why such a mutation would result in an inactive
enzyme.
4. Osteoclasts in bone tissue are particularly rich in carbonic anhydrase II, and a proper functioning
enzyme is critical to the development of healthy tissue. In order for proper bone development to
occur, the osteoclast must acidify the bone-resorbing compartment. Also involved in this acidification
are several transporters: a Na+/H+ exchanger, a Cl-/HCO3- exchanger and the Na+K+ATPase, which
exchanges Na+ and K+ ions. (An exchanger is a protein or protein complex located in the cell
membrane which transports one ion in one
direction and the second ion in the other
direction simultaneously.)
A partial diagram of the osteoclast is
shown in Figure 3.1. Fill in the blanks in
the diagram indicating the roles of
carbonic anhydrase II and the exchangers
in the acidification of the bone-resorbing
compartment. Include the reactants and
products of the appropriate intracellular
reaction(s) and note in which direction
each ion is transported in the osteoclast.
Figure 3.1: The role of the osteoclast intracellular carbonic
anhydrase II in establishing the acidity of the boneresorbing compartment.

References
Sly, W. S., and Hu, P. Y. (1995) Ann. Rev. Biochem., 64, pp. 375-401.
Whyte, M. P. (1993) Clin. Orthop. Relat. Res., 294, pp. 52-63.
2

Case 13
Inhibition of Alcohol Dehydrogenase
Focus concept
The inhibition of the alcohol dehydrogenase by a formamide compound is examined.

Prerequisites
C
C

Principles of enzyme kinetics
Identification of inhibition via Lineweaver-Burk plots

Background
Alcohol dehydrogenase (ADH) is the enzyme that is responsible for converting ethanol to
acetaldehyde (the reaction is shown in Figure 13.1). It is the enzyme responsible for the
metabolism of ethanol in the alcoholic beverages we consume. Five different isozymes of ADH
have been identified, and it has been shown that the enzyme has a rather broad substrate
specificity and can oxidize aldehydes as well as primary and secondary alcohols. For example,
ADH can also oxidize methanol (wood alcohol) and ethylene alcohol (antifreeze). The poisonous
nature of these compounds results from the ADH-catalyzed conversion of these compounds to
toxic products. For example, ADH converts methanol to formaldehyde, which is toxic to the
optic nerve and can produce blindness. In high doses, formaldehyde may be fatal.
In this study, the authors investigated the ability of formamide compounds to inhibit alcohol
dehydrogenase. Only a portion of their data is presented here. The authors were able to propose
a mechanism for the inhibition from the extensive data they collected using a wide variety of
formamide compounds. The mechanism is shown in Figure 13.2.

Figure 13.1: ADH-catalyzed oxidation of ethanol.

1

Figure 13.2: Mechanism of ADH1. The inhibitor binds as an
aldehyde analog.

Questions
1. Certain individuals are more sensitive to alcohol than others. For example, women are more
sensitive to alcohol than men–even when body weight and % body fat are taken into account,
women become more intoxicated than men consuming an identical amount of alcohol. Using
what we have learned in the enzyme chapters, give biochemical reasons that would explain
why women become more intoxicated than men when consuming an equal amount of alcohol.
2. A treatment for methanol poisoning is to have the victim drink large amounts of ethanol. Why
might this be an effective treatment?
3. The authors of this study studied the ability of N-1,5-dimethylhexylformamide to inhibit mouse
ADH1. The activity of the enzyme was measured in the absence of inhibitor, and in the
presence of 1.0 :M inhibitor. The data are presented in Table 13.1.
Table 13.1: Inhibition of mouse ADH1 by N,1-5-dimethylhexylformamide

Ethanol
Concentration, mM

ADH1 velocity, ) NADH
absorbance/min (without
inhibitor)

ADH1 velocity, ) NADH
absorbance/min (with
inhibitor)

0.20

0.036

0.022

0.25

0.042

0.024

0.36

0.048

0.027

0.60

0.065

0.029

2.00

0.075

0.033
2

a. What are the KM and Vmax values for ADH in the absence of inhibitor? in the presence of
the inhibitor?
b. What type of inhibitor is N-1,5-dimethylhexylformamide? Explain.
c. Calculate the values of " and/or "’, if they are significantly different from 1. What kind of
inhibitor is N-1,5-dimethylhexylformamide? Explain.
d. Calculate the Ki and/or Ki’ (whichever is appropriate) for N-1,5-dimethylhexylformamide
(Hint: You can obtain these values from " and "’).
4. The authors describe the mechanism of ADH as an “ordered bi-bi” mechanism. Give a written
description of the mechanism, as shown in Figure 13.2. How does N-1,5dimethylhexylformamide inhibit the activity of the ADH enzyme? How does N-1,5dimethylhexylformamide differ from the “classic” inhibitors of this type that are described in
our textbook?
5. The authors found that a class of compounds called pyrazoles were also inhibitors of ADH.
These inhibitors bind to the E-NAD+ complex. What kind of inhibitor are pyrazoles? Are
these inhibitors the same or different as the formamides?
6. a. Would N-1,5-dimethylhexylformamide be an effective alternative for the treatment of
methanol and ethylene glycol poisoning, assuming that it is non-toxic itself (and as an
alternative to getting the patient drunk, as described in Question 2)? Would N-1,5dimethylhexylformamide be effective even if the concentrations of methanol or ethylene
glycol were very high? (Hint: Compare the values of KI or KI’, whichever is appropriate,
and KM).
b. The compound 4-methyl pyrazole is currently being used as a treatment for methanol
poisoning. How would the effectiveness of 4-methyl pyrazole compare with the
effectiveness of a formamide treatment?

Reference
Venkataramaiah, T. H., and Plapp, B. V. (2003) J. Biol. Chem. 278, pp. 36699-36706.

3

Case 20
NAD+-dependent Glyceraldehyde-3-phosphate
Dehydrogenase from Thermoproteus tenax
Focus concept
Glycolytic enzymes from Thermoproteus tenax are regulated in an unusual manner.

Prerequisites
C
C
C

The glycolytic pathway.
Enzyme kinetics and inhibition.
The cooperative nature of regulated enzymes.

Background
Carbohydrate metabolism in the thermophilic archaeal bacterium Thermoproteus tenax is rather
peculiar compared to the types of organisms usually studied in introductory biochemistry. For example,
the phosphofructokinase reaction in T. tenax is reversible, and is dependent upon pyrophosphate rather
than ATP. In addition, T. tenax has two different glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
isoenzymes. One is well known and, although it requires NADP+ as a cofactor instead of NAD+, it
resembles the GAPDH enzyme we studied in class and is referred to as the “phosphorylating GAPDH”.
In contrast, the second isoenzyme is irreversible and requires NAD+ as a cofactor and is referred to as the
“nonphosphorylating GAPDH”. In this case, we will consider the properties of the latter enzyme. The
balanced equation of the reaction catalyzed by the nonphosphorylating NAD+-dependent glyceraldehyde3-phosphate dehydrogenase is shown below.

Figure 20.1: Non-phosphorylating NAD+-dependent GAPDH in T. tenax.

T. tenax stores energy in the form of glycogen, which is degraded to glucose-1-phosphate. The
glucose-1-phosphate is then converted to glucose-6-phosphate and then enters the glycolytic pathway.
The two GAPDH enzymes are probably differently regulated in T. tenax. The authors of this study
propose that “phosphorylating, NADP+-dependent” GAPDH is involved in efficient ATP production
whereas the “non-phosphorylating, NAD+-dependent” GAPDH is somewhat involved in ATP production
but is also involved in providing intermediates for cellular biosynthetic reactions.
1

CASE 20 C NAD+-dependent Glyceraldehyde-3-phosphate Dehydrogenase from Thermoproteus tenax
The gene for the non-phosphorylating, NAD+-dependent GAPDH was cloned and sequenced and its
kinetic characteristics were studied. Summary information is presented in Table 1.1.
Table 20.1: Kinetic properties of NAD+-dependent GAPDH isolated from T. tenax.
NAD+ saturation
Without AMP
Vmax, units/mg
KM, mM
With AMP
Vmax, units/mg
KM, mM

36.5
3.3
37.0
1.4

Molecular Mass
Subunit (kD)
Native (kD)

55,000
220,000

Questions
1. Name the three enzymes that catalyze irreversible, regulated reactions in glycolysis as studied in class.
2. What is the significance of the GAPDH reaction (in E. coli, the enzyme discussed in class) to
glycolysis?
3. How does the reaction catalyzed by GAPDH from T. tenax presented here differ from the reaction
carried out in E. coli (ie, the reaction discussed in class)?
4. The activity of the GAPDH enzyme was assayed in the presence of a constant amount of
glyceraldehyde-3-phosphate and an increasing amount of NAD+. The activity of the control was
compared to the activity in the presence of various metabolites. The results are shown in Figure 20.2.
Additional data are given in Table 20.2.
a. Use the data in Figure 20.2 to estimate a KM value for the enzyme in the presence of these
metabolites. Classify the metabolites listed in Table 20.2 as inhibitors or activators. Fill in your
answers in the table provided. Explain how you decided whether these metabolites are inhibitors
or activators, based on the graph.
b. How would you classify NADH, ADP and ATP? (These data are not presented in the graph). Are
they inhibitors or activators? Add this information to Table 20.2.
c. Explain the physiological significance of your answers to questions 4a and 4b.

2

CASE 20 C NAD+-dependent Glyceraldehyde-3-phosphate Dehydrogenase from Thermoproteus tenax
Table 20.2: Effect of various metabolites on the activity of NAD+-dependent GAPDH isolated from T. tenax.
(Based on Brunner, et al., 1998.)

Metabolite

Apparent KM, mM

Inhibitor or activator?

None
NADP+
Glucose-1-phosphate
AMP
NADH
ATP
ADP
5. The Hill coefficients for NAD+ binding to the T. tenax GADPH in the presence and absence of
NADP+ were measured and are shown on the graph in Figure 20.2. What is the significance of the
change in the value of the Hill coefficient? Is this consistent with (a) the shape of the curve and (b)
the information given in the background on the enzyme’s structure?
6. What is the ATP yield for one mole of
glucose oxidized by the pathway that
uses the nonphosphorylating GAPDH
enzyme?

Reference
Brunner, N. A., Brinkmann, H., Siebers,
B., and Hensel, R. (1998) J. Biol. Chem.
273, pp. 6149-6156.

Figure 20.2: Cosubstrate saturation of NAD+-dependent
GAPDH of T. tenax in the presence of various effects.
Assay conditions were the following: 90 mM HEPES, pH
= 7, 160 mM KCl, 4 mM D L -GAP (based on Brunner, et
al., 1998)