Importance of Hydrogen-Bond Formation in Stabilizing the Transition State of Subtilisin

Structural studies on serine proteinases have shown that hydrogen bonds are involved in stabilizing the charged tetrahedral intermediate in the transition-state complex. However, little is known about the quantitative contribution of these interactions to transition-state stabilization. X-ray crystallographic studies of subtilisin (Robertus, J. D., Kraut, J., Alden, R. A. & Birktoft, J. J. Biochemistry, Wash. 11, 4293-4303 (1972)) have suggested that the amide side chain from asparagine-155 forms a hydrogen bond with the oxyanion produced on the substrate carbonyl oxygen in the tetrahedral intermediate. To study the importance of the Asn-155 hydrogen bond in stabilizing the tetrahedral intermediate, Asn-155 was substituted with Thr, His, Gln and Asp by using site-specific mutagenesis of the cloned subtilisin gene from B. amyloliquefaciens. These substitutions were intended to alter the position and charge of the potential hydrogen-bonding group at 155. Mutations of Asn-155 caused large decreases in substrate turnover, k_cat (200- to 4000-fold), with marginal decreases in substrate binding, K_M (up to 7-fold). The most dramatic effects were seen with Thr-155, where k_cat was reduced 4000-fold with a slight increase in K_M. Mutations of Asn-155 caused a loss in transition-state stabilization energy of 9.2-20 kJ mol^-1 Simple enrichment methods are described which greatly facilitate the isolation of mutant sequences. These methods depend upon the introduction or elimination of a unique and silent restriction site near the site of mutagenesis.