Bastyns K, Froeyen M, Diaz J F, Volckaert G, Engelborghs Y
Laboratory of Chemical and Biological Dynamics, University of Leuven, Belgium.
Proteins. 1996 Mar;24(3):370-8. doi: 10.1002/(SICI)1097-0134(199603)24:3<370::AID-PROT10>3.0.CO;2-J.
Barnase, the guanine specific ribonuclease of Bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme. Ser-85 was mutated into Glu with the goal to introduce an extra charge in the neighborhood of His-102. A double mutation (Ser-85-Glu and Asp-86-Asn) was introduced with the same purpose but without altering the global charge of the enzyme. A similar set of mutations was made using Asp at position 85. For all mutants the pI was determined using the technique of isoelectric focusing and calculated on the basis of the Tanford-Kirkwood theory. When Glu was used to replace Ser-85, the correlation between the experimental and the calculated values was perfect. However, in the Ser-85-Asp mutant, the experimental pI drop is bigger than the calculated one, and in the double mutant (Ser-85-Asp and Asp-86-Asn) the compensation is not achieved. The effect of the mutations on the pKa of His-102 can be determined from the pH dependence of the kcat/KM for the hydrolysis of dinucleotides, e.g., GpC. The effect can also be calculated using the the method of Honig. In this case the agreement is very good for the Glu-mutants and the single Asp-mutant, but less for the double Asp-mutant. The global stability of the Asp-mutants is, however, the same as the wild type, as shown by stability studies using urea denaturation. Molecular dynamics calculations, however, show that in the double Asp-mutant His-102 (H+) swings out of its pocket to make a hydrogen bridge with Gin-104 which should cause an additional pKa rise. The effect of the Glu-mutations was also tested on all the kinetic parameters for GpC and the cyclic intermediate G > p at pH 6.5, for RNA at pH 8.0, and for poly(A) at pH 6.2. The effect of the mutations is rather limited for the dinucleotide and the cyclic intermediate, but a strong increase of the KM is observed in the case of the single mutant (extra negative charge) with polymeric substrates. These results indicate that the extra negative charge has a strong destabilizing effect on the binding of the polymeric substrates in the ground state and the transition state complex. A comparison with the structure of bound tetranucleotides (Buckle, A.M. and Fersht, A.R., Biochemistry 33:1644-1653, 1994) shows that the extra negative charge points towards the P2 site.
芽孢杆菌核糖核酸酶Barnase是解淀粉芽孢杆菌的鸟嘌呤特异性核糖核酸酶，为改变该酶的静电性质，对其进行了突变。将Ser-85突变为Glu，目的是在His-102附近引入一个额外电荷。为达到相同目的但不改变酶的整体电荷，引入了双重突变（Ser-85-Glu和Asp-86-Asn）。在85位使用Asp进行了类似的一组突变。对于所有突变体，使用等电聚焦技术测定其pI，并根据Tanford-Kirkwood理论进行计算。当用Glu取代Ser-85时，实验值与计算值之间的相关性极佳。然而，在Ser-85-Asp突变体中，实验测得的pI下降幅度大于计算值，而在双重突变体（Ser-85-Asp和Asp-86-Asn）中，电荷补偿未实现。His-102的pKa的突变效应可通过二核苷酸（如GpC）水解的kcat/KM对pH的依赖性来确定。该效应也可使用Honig方法进行计算。在这种情况下，对于Glu突变体和单个Asp突变体，二者吻合度很高，但对于双重Asp突变体，吻合度较低。然而，如使用尿素变性的稳定性研究所示，Asp突变体的整体稳定性与野生型相同。不过，分子动力学计算表明，在双重Asp突变体中，His-102（H+）从其口袋中摆动出来，与Gin-104形成氢键，这应会导致pKa额外升高。还在pH 6.5下对GpC和环状中间体G > p的所有动力学参数、pH 8.0下对RNA以及pH 6.2下对聚腺苷酸测试了Glu突变的影响。对于二核苷酸和环状中间体，突变的影响相当有限，但在具有聚合物底物的单个突变体（额外负电荷）情况下，观察到KM大幅增加。这些结果表明，额外的负电荷对聚合物底物在基态和过渡态复合物中的结合具有很强的去稳定作用。与结合的四核苷酸结构（Buckle, A.M.和Fersht, A.R., Biochemistry 33:1644 - 1653, 1994）的比较表明，额外的负电荷指向P2位点。
