Specific DNA Recognition by the Type II Restriction Endonuclease MunI: The Effect of pH
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- 作者：Ihtshamul Haq ; Ronan O'Brien ; Arunas Lagunavicius ; Virginijus Siksnys ; and John E. Ladbury
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：December 11, 2001
- 年：2001
- 卷：40
- 期：49
- 页码：14960 - 14967
- 全文大小：89K
- 年卷期：v.40,no.49(December 11, 2001)
- ISSN：1520-4995
文摘
To investigate the effect of pH on sequence-specific binding, a thermodynamic characterizationof the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed.MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activityin the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25
C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constantpressure heat capacity. On going through the pH range 5.75-9.0 at 25 C, the affinity of specific complexformation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed tobe on the protein. Based on the simplest model that will fit the data, two distinct protonation events areobserved. At low pH, two groups per protein molecule undergo protonation with a pKa of 6.0 and 6.9 inthe free and bound forms, respectively. At high pH, a further independent protonation occurs involvingtwo groups with pKa values of 8.9 and approximately 10.7 in the free and bound forms, respectively. Thechange in heat capacity ranges from -2.7 to -1.7 kJ mol-1 K-1 in going from pH 6.5 to 8.5. This rangeof variation of change in heat capacity can be accounted for by the effects of protonation of the interactingmolecules. The change in heat capacity, calculated from surface area burial using a previously establishedrelationship (1.15 kJ mol-1 K-1), does not correlate well with the experimentally determined values.
C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constantpressure heat capacity. On going through the pH range 5.75-9.0 at 25 C, the affinity of specific complexformation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed tobe on the protein. Based on the simplest model that will fit the data, two distinct protonation events areobserved. At low pH, two groups per protein molecule undergo protonation with a pKa of 6.0 and 6.9 inthe free and bound forms, respectively. At high pH, a further independent protonation occurs involvingtwo groups with pKa values of 8.9 and approximately 10.7 in the free and bound forms, respectively. Thechange in heat capacity ranges from -2.7 to -1.7 kJ mol-1 K-1 in going from pH 6.5 to 8.5. This rangeof variation of change in heat capacity can be accounted for by the effects of protonation of the interactingmolecules. The change in heat capacity, calculated from surface area burial using a previously establishedrelationship (1.15 kJ mol-1 K-1), does not correlate well with the experimentally determined values.