Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E. T. (1987) J. Am. Chem. SOC. 109, 3808-38101. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S22 1C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221 C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S22 1C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose a-amino group attacks the thioacyl-enzyme.To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S22 1 C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P, residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants. The set of specific peptide ligases should be useful for blockwise synthesis or semisynthesis of proteins in aqueous solution.C h e m i c a l approaches for synthesis and engineering of proteins offer many advantages to recombinant methods in that one can incorporate nonnatural or selectively labeled amino acids. However, peptide synthesis is practically limited to small proteins (typically <50 residues) due to the accumulation of side products and racemization that complicate product purification and decrease yields [for recent reviews see Kaiser (1989) and Offord (1987)l.Proteolytic enzymes, in particular serine proteases, have been used as alternatives to synthetic peptide chemistry because of their stereoselective properties and mild reaction conditions [for reviews see Kullmann (1987) and Chaiken (1981)l. Such enzymes have also been used to complement chemical coupling methods and allow larger proteins to be synthesized by blockwise enzymatic coupling of synthetic fragments [Tnouye et al., 1979; for review see Chaiken (1981)l. However, the narrow substrate specificities and hydrolytic activities of serine proteases have limited their use in peptide synthesis.A central problem in the use of serine proteases for peptide synthesis is that hydrolysis of the acyl-enzyme intermediate is strongly favored over aminoly...
This investigation was intended to determine whether students with learning disabilities (LD) and mild mental retardation (MR) differed from normally achieving students with respect to inductive thinking on an inquiry learning task involving pendulum motion. Twenty normally achieving students, 18 students with LD, and 16 students with MR were provided individually with guided coaching, in a prespecified sequence of steps, intended to promote induction of the association between pendulum length and pendulum motion. After the initial guided activity, 75% of the normally achieving students and 50% of the students with LD, but none of the students with MR, provided the correct induction. Over one fourth of the students with LD, and nearly all of the students with MR, required all of the prescribed coaching levels, including direct explanation of the rule. Students with LD and MR were less likely to correctly answer transfer/application questions. Implications for teaching are provided.
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