Frandsen T P, Christensen T, Stoffer B, Lehmbeck J, Dupont C, Honzatko R B, Svensson B
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Biochemistry. 1995 Aug 15;34(32):10162-9. doi: 10.1021/bi00032a009.
The mutants Arg54-->Leu, Arg54-->Lys, Arg305-->Lys, Asp309-->Glu, and Trp317-->Phe, located at subsites 1 and 2 in glucoamylase from Aspergillus niger, provide insight into the importance of specific hydrogen bonds and hydrophobic interactions in substrate recognition, catalytic mechanism, and stability. As suggested from the crystal structure of a closely related glucoamylase [Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631-15639], Arg54 in subsite 1 hydrogen bonds to the key polar group 4'-OH of maltose. The two mutants of Arg54 display losses in transition-state stabilization of 16-21 kJ mol-1 in the hydrolysis of different maltooligodextrins, which originate from a [(1.2-1.8) x 10(3)]-fold reduction in kcat and changes in Km ranging from 25% to 300% of the wild-type values. Arg305 similarly hydrogen bonds to 2'-OH and 3-OH, located at subsites 1 and 2, respectively. Arg305-->Lys glucoamylase is not saturated at concentrations of maltose or maltoheptaose of 400- and 40-fold, respectively, the Km of the wild-type enzyme. This mutant also has highly reduced kcat. On the other hand, for the alpha-1,6-linked isomaltose, the Lys305 mutant surprisingly has the same Km as the wild-type enzyme, while kcat is 10(3)-fold reduced. Arg305 is thus an important determinant in the distinction of the alpha-1,4 to alpha-1,6 substrate specificity. Arg305 interacts electrostatically and hydrophobically with the side chains of Asp309 and Trp317.(ABSTRACT TRUNCATED AT 250 WORDS)
位于黑曲霉葡糖淀粉酶亚位点1和2的突变体Arg54→Leu、Arg54→Lys、Arg305→Lys、Asp309→Glu和Trp317→Phe，有助于深入了解特定氢键和疏水相互作用在底物识别、催化机制及稳定性方面的重要性。正如一种密切相关的葡糖淀粉酶的晶体结构所表明的那样[Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631 - 15639]，亚位点1中的Arg54与麦芽糖的关键极性基团4'-OH形成氢键。在不同麦芽寡糖的水解过程中，Arg54的两个突变体在过渡态稳定化方面损失了16 - 21 kJ mol-1，这源于kcat降低了[(1.2 - 1.8)×10(3)]倍，且Km变化范围为野生型值的25%至300%。Arg305同样分别与位于亚位点1和2的2'-OH和3-OH形成氢键。Arg305→Lys葡糖淀粉酶在麦芽糖或麦芽七糖浓度分别为野生型酶Km的400倍和40倍时未达到饱和。该突变体的kcat也大幅降低。另一方面，对于α-1,6-连接的异麦芽糖，Lys305突变体的Km与野生型酶相同，而kcat降低了10(3)倍。因此，Arg305是区分α-1,4与α-
