Just an additional hydrogen bond can dramatically reduce the catalytic activity of Bacillus subtilis lipase A I12T mutant: An integration of computational modeling and experimental analysis

Understanding the structural basis and energetic property of hydrogen bonding and its effects on enzymatic activity is fundamentally important for the rational design of specific enzymes with desired biological functions. In the current study, site-directed mutagenesis analysis preliminarily revealed that the amino acid substitution of Ile12 with Thr12 (I12T) dramatically reduced the hydrolytic activity of Bacillus subtilis lipase A. A further computational investigation proposed that the I12T mutation would establish a geometrically perfect hydrogen bond between the mutated Thr12 and catalytic Ser77 of lipase A, which considerably impaired the catalytic capability of lipase A through two distinct but complementary approaches: rigidizing the enzyme active site and lowering the nucleophilic ability of the catalytic residue Ser77. To verify this hypothesis, a homogenous mutation I12S serving as the control to the I12T mutation was created to examine the hydrogen bonding effect on enzymatic activity. It was found that the I12S mutant only suffered from a slight damage in its hydrolytic ability due to absence of the hydrogen bond originally present at the Thr12-Ser77 interface in the I12T mutant, which was further characterized systematically by quantum mechanics/molecular mechanics (QM/MM) modeling, atom-in-molecules (AIM) analysis and molecular dynamics (MD) simulation. It is suggested that the hydrogen bond arising from the I12T mutation in lipase A can considerably reduce the flexibility and mobility of the enzyme active site, thus impairing the catalytic activity of the lipase A I12T mutant remarkably; the activity loss can be, however, largely recovered by replacing Thr residue at the 12th position of I12T mutant with its analog Ser, which is chemically similar to Thr but cannot form effective hydrogen bonding with Ser77.