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The Catalytic Site of Lysozyme


Parsons, Stanley Monroe (1971) The Catalytic Site of Lysozyme. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/P8Q0-TK36.


Proton magnetic resonance techniques have used to study the association of simple cyclohexane derivatives to the primary binding subsite of lysozyme in solution. The results agree in detail with crystallographic deductions about the nature of the important binding interactions in subsite C.

A reagent new to protein modification studies, triethyloxonium fluoroborate, has been shown to selectively esterify carboxyl groups under mild aqueous conditions. Two single ethylesters of lysozyme were isolated chromatographically in good yield and were characterized. One is enzymatically active and reverts to lysozyme under neutral conditions. The other is enzymatically inactive but still binds the inhibitor chitotriose. This inactive ester was identified as occurring at aspartic acid residue 52 by α-chymotryptic digestion of the oxidized derivative and direct observation of the β-ethyl aspartate released from the modified peptide by carboxypeptidase.

Difference pH titrations of the Asp 52 ester vs. the native lysozyme show a difference in proton uptake due to one group over the pH range 3 to 9. The titration of glutamic acid residue 35 in the derivative is perturbed, though, and from the shape of the difference titration curve the ionization behavior of Asp 52 and Glu 35 in native lysozyme and of Glu 35' in the derivative can be determined. Four microscopic constants for the interacting Asp 52-Glu 35 system considered as a dibasic acid are calculated for each difference curve. The macroscopic pK of Asp 52 is 4.5 and that of Glu 35 is 5.9 in 0.15 M KCl at 25° C. The ionization of Asp 52 is very dependent on the ionic strength of the solution in going from 0.02 M to 0.50 M KCl and it is shown that Asp 52 is in an environment only somewhat less hydrophobic than that of Glu 35. The heats of ionization determined over the temperature range 1.6° to 40° give an observed ΔH equal to 3.5 kcal/mole for Asp 52 and 2.9 kcal/mole for Glu 35. When these values are corrected for charge interactions in an approximate way they become more normal. The corrected value for Asp 52 can be interpreted in a manner consistent with hydrogen bonding to Asn 46 and Asn 59.

Lysozyme inhibitors and small substrates are found not to perturb the ionization of Asp 52. The macroscopic pK of Glu 35 increases to 6.5 when binding subsites A through D are filled. The pK of Glu 35 exhibits a dramatic shift to 8-8.5 in the presence of the high molecular weight substrate glycol chitin. In addition, the pK of Asp 101 is determined to be about 4.1 in free lysozyme, about 3.9 when subsite B is filled, and about 3.7 when subsite A is filled. These hydrogen bond mediated pK changes do not occur in the derivative and apparently require a precise binding orientation.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:(Chemistry)
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Raftery, Michael A.
Thesis Committee:
  • Unknown, Unknown
Defense Date:31 July 1970
Funding AgencyGrant Number
Record Number:CaltechTHESIS:04052018-084556110
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10791
Deposited By: Benjamin Perez
Deposited On:05 Apr 2018 18:09
Last Modified:18 Jul 2023 23:06

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