Klein R R, King G, Moreau R A, Haas M J
ERRC, ARS, USDA, Wyndmoor, Pennsylvania 19038, USA.
Lipids. 1997 Feb;32(2):123-30. doi: 10.1007/s11745-997-0016-1.
The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short- or medium-chain length fatty acids. Computer-generated structural models of R. delemar lipase were used in mutant protein design and in the interpretation of the catalytic properties of the resulting recombinant enzymes. Molecular dynamics simulations of the double mutant, val209trp + phe112trp, predicted that the introduction of trp112 and trp209 in the acyl binding groove would sterically hinder the docking of fatty acids longer than butyric acid. Assayed against a mixture of triacylglycerol substrates, the val209trp + phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributyrin relative to the hydrolysis of tricaprylin while no triolein hydrolysis was detected. By comparison, the val94Trp mutant, predicted to pose steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fold increase in tricaprylin hydrolysis relative to the hydrolysis of tributyrin. Molecular models of the double mutant phe95asp + phe214arg indicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mixture of triacylglycerols, the phe95asp + phe214arg substitutions resulted in an enzyme with 3-fold enhanced relative activity for tricaprylin compared to triolein, suggesting that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 A of the active site by the double mutation leu146lys + ser115asp destroyed catalytic activity entirely. Similarly, the substitution of polar Gln at the rim of the acyl binding groove for phe112 largely eliminated catalytic activity of the lipase.
通过定点诱变改变了德氏根霉前脂肪酶和成熟脂肪酶的酰基结合位点，以提高脂肪酶对短链或中链脂肪酸的特异性。德氏根霉脂肪酶的计算机生成结构模型用于突变蛋白设计以及对所得重组酶催化特性的解释。双突变体val209trp + phe112trp的分子动力学模拟预测，在酰基结合槽中引入trp112和trp209会在空间上阻碍比丁酸更长的脂肪酸对接。针对三酰甘油底物混合物进行测定时，val209trp + phe112trp成熟脂肪酶突变体相对于三辛酸甘油酯的水解，三丁酸甘油酯的水解增加了80倍，而未检测到三油酸甘油酯的水解。相比之下，预测val94Trp突变体在酰基结合槽中对接比辛酸更长的脂肪酸会产生空间或几何限制，导致三辛酸甘油酯的水解相对于三丁酸甘油酯的水解适度增加了1.4倍。双突变体phe95asp + phe214arg的分子模型表明，在酰基结合槽远端的asp95和arg214之间形成了盐桥。当用三酰甘油混合物进行检测时，phe95asp + phe214arg替换产生的酶对三辛酸甘油酯的相对活性比三油酸甘油酯提高了3倍，这表明中链长度特异性的结构决定因素可能存在于酰基结合槽的远端。通过leu146lys + ser115asp双突变在活性位点8埃范围内引入盐桥的尝试完全破坏了催化活性。同样，用酰基结合槽边缘的极性Gln取代phe112在很大程度上消除了脂肪酶的催化活性。
