Recitation #6: Enzyme Mechanisms and Regulation
Case Study:
Nonenzymatic Deamidation of Asparagine and Glutamine
Residues in Proteins
Background:
Asparagine (Asn) and glutamine (Gln) residues in proteins will sometimes be
nonenzymatically hydrolytically deamidated to aspartate (Asp) and glutamate (Glu)
residues, respectively. An exhaustive study of proteins known to undergo
deamidation has revealed that the rate of deamidation is influenced by the amino
acids preceding and following the amide amino acids in primary sequence. Other
amino acids that might be far apart in primary sequence to the deamidated amino
acids, but close in terms of tertiary structure, might also influence the deamidation
process.
The extent to which deamidation occurs might be underestimated. Proteins
purified using traditional biochemical techniques frequently provide an
environment in which deamidation can occur readily. Since Asp and Glu residues
resulting from deamidation are indistinguishable from Asp and Glu residues
originally present in the protein, the proper sequence of the protein is not always
known. Thus, the only way to know whether a protein undergoes deamidation is to
compare the sequences of bases in the gene to the amino acid sequence of the
purified protein. Unfortunately, these data are not available for every protein that
undergoes deamidation.
The deamidation of amide side chains results in a change in the tertiary
structure of the protein. In fact, it has been suggested that deamidation actually
functions as a molecular timer for protein turnover, since it has been observed that
deamidation of some proteins increases their susceptibility to degradation by
proteases. Proteins that turn over rapidly might do so as a result of a rapid
deamidation process which would signal cellular proteases to destroy the
deamidated protein. In the same vein, deamidation might be involved in the aging
process. This hypothesis is best tested in proteins such as the lens crystallin protein
that turn over slowly or not at all. It has been shown in chickens that 15% of the lens
crystallin protein is deamidated at age four months, whereas 50% of the protein is
deamidated at one year and 70% at ten years.
, Case Study Questions:
1. Asparagine and glutamine residues in proteins are deamidated in an aqueous
solution to yield aspartate and glutamate, respectively, and ammonium. Write
the net reaction for asparagine deamidation.
O O
NH CH C +
NH CH C + H 2O + NH4
CH2 CH2
C O C O
NH2 O-
Asparagine Aspartate
2. Proteins undergoing deamidation of one or more of their Asn or Gln groups have
been detected by isoelectric focusing. Why would isoelectric focusing be
effective in separating deamidated from non-deamidated protein? Compare the
relative pI values of the deamidated and non-deamidated proteins.
Asparagine and glutamine have neutral amino acid side chains whereas aspartate and
glutamate side chains are negatively charged. Thus, it would be possible to detect
deamidated proteins because they would have a more acidic (lower) pI value than
non-deamidated proteins.
3. Explain how deamidation could affect the tertiary structure of a protein. Be
specific about what kinds of non-covalent interactions might be involved.
Deamidation produces negatively charged amino acid side chains which could form ion
pairs with nearby positively charged amino acid side chains, thus altering the three-
dimensional conformation of the protein. Negatively charged amino acids would also
repel any other nearby negative charges. Asn and Gln are neutral and cannot form ion
pairs. The conversion from an amide to carboxylic acid functional group also changes the
hydrogen bonding capability of the side chains. The amide side chain can serve as both
hydrogen bond donor and acceptor whereas the carboxylic acid group can only serve as a
hydrogen bond acceptor.
Case Study:
Nonenzymatic Deamidation of Asparagine and Glutamine
Residues in Proteins
Background:
Asparagine (Asn) and glutamine (Gln) residues in proteins will sometimes be
nonenzymatically hydrolytically deamidated to aspartate (Asp) and glutamate (Glu)
residues, respectively. An exhaustive study of proteins known to undergo
deamidation has revealed that the rate of deamidation is influenced by the amino
acids preceding and following the amide amino acids in primary sequence. Other
amino acids that might be far apart in primary sequence to the deamidated amino
acids, but close in terms of tertiary structure, might also influence the deamidation
process.
The extent to which deamidation occurs might be underestimated. Proteins
purified using traditional biochemical techniques frequently provide an
environment in which deamidation can occur readily. Since Asp and Glu residues
resulting from deamidation are indistinguishable from Asp and Glu residues
originally present in the protein, the proper sequence of the protein is not always
known. Thus, the only way to know whether a protein undergoes deamidation is to
compare the sequences of bases in the gene to the amino acid sequence of the
purified protein. Unfortunately, these data are not available for every protein that
undergoes deamidation.
The deamidation of amide side chains results in a change in the tertiary
structure of the protein. In fact, it has been suggested that deamidation actually
functions as a molecular timer for protein turnover, since it has been observed that
deamidation of some proteins increases their susceptibility to degradation by
proteases. Proteins that turn over rapidly might do so as a result of a rapid
deamidation process which would signal cellular proteases to destroy the
deamidated protein. In the same vein, deamidation might be involved in the aging
process. This hypothesis is best tested in proteins such as the lens crystallin protein
that turn over slowly or not at all. It has been shown in chickens that 15% of the lens
crystallin protein is deamidated at age four months, whereas 50% of the protein is
deamidated at one year and 70% at ten years.
, Case Study Questions:
1. Asparagine and glutamine residues in proteins are deamidated in an aqueous
solution to yield aspartate and glutamate, respectively, and ammonium. Write
the net reaction for asparagine deamidation.
O O
NH CH C +
NH CH C + H 2O + NH4
CH2 CH2
C O C O
NH2 O-
Asparagine Aspartate
2. Proteins undergoing deamidation of one or more of their Asn or Gln groups have
been detected by isoelectric focusing. Why would isoelectric focusing be
effective in separating deamidated from non-deamidated protein? Compare the
relative pI values of the deamidated and non-deamidated proteins.
Asparagine and glutamine have neutral amino acid side chains whereas aspartate and
glutamate side chains are negatively charged. Thus, it would be possible to detect
deamidated proteins because they would have a more acidic (lower) pI value than
non-deamidated proteins.
3. Explain how deamidation could affect the tertiary structure of a protein. Be
specific about what kinds of non-covalent interactions might be involved.
Deamidation produces negatively charged amino acid side chains which could form ion
pairs with nearby positively charged amino acid side chains, thus altering the three-
dimensional conformation of the protein. Negatively charged amino acids would also
repel any other nearby negative charges. Asn and Gln are neutral and cannot form ion
pairs. The conversion from an amide to carboxylic acid functional group also changes the
hydrogen bonding capability of the side chains. The amide side chain can serve as both
hydrogen bond donor and acceptor whereas the carboxylic acid group can only serve as a
hydrogen bond acceptor.
