See the Figure in Problem 20 for the structures and 3-letter abbreviations of amino acids. Subtilisi — Organic Chemistry Chemistry Question
Substrate Specificity of Subtilisin
See the Figure in Problem 20 for the structures and 3-letter abbreviations of amino acids.
Subtilisin is a serine protease produced by the bacterium Bacillus amyloliquefasciens that catalyzes hydrolysis of peptide bonds in proteins:
[VISUAL]
More generally, serine proteases catalyze transfer of an acyl group from a donor molecule such as an amide or ester R(C=O-L, to an acceptor nucleophile (Nuc) such as water:
[VISUAL]
The Figure below shows a schematic of a peptide substrate bound in the active site of subtilisin (the gray surface represents the enzyme itself). Ser221 and His64 are two amino acid residues in the active site that are essential for catalysis of peptide bond hydrolysis.
[VISUAL]
Subtilisin has an extensive substrate binding site in which are bound four amino acid residues on the N-terminal side of the peptide bond that is hydrolyzed. The side chains of these four residues are bound in four “subsites” in the enzyme, called S1–S4. Amino acid residues of subtilisin whose side chains project into the sub-sites are indicated in the Figure above: Gly166 in subsite S1, Asn62 in subsite S2, and Tyr104 in subsite S4. The chemical and structural properties of these residues from the enzyme determine which peptide substrates are bound and hydrolyzed by the subtilisin.
The peptide-p-nitroanilide substrate with the sequence: Ala-Ala-Pro-Phe- p-nitroanilide is hydrolyzed rapidly by subtilisin because the four amino acid residues in the substrate fit well into the binding sub-sites (the Ala-Ala-Pro-Phe residues are bound in subsites S4–S1, respectively).
Site-directed mutagenesis can be used to change residues in the binding subsites of subtilisin to alter the substrate specificity of the enzyme. In one experiment, Gly166 was changed to Ile (Gly166Ile mutant) and the catalytic activity of the mutant enzyme was tested with the following peptide substrates:
I Ala-Ala-Pro-Phe-p-nitroanilide
II Ala-Ala-Pro-Ala-p-nitroanilide
III Ala-Ala-Pro-Glu-p-nitroanilide
IV Ala-Ala-Pro-Tyr-p-nitroanilide
Which peptide would be hydrolyzed most rapidly (highest kcat/Km) by the Gly166Ile mutant enzyme?
Model Answer
The best substrate is II.
The mutation changes an amino acid with a very small side chain (glycine) to a much larger (and non-polar) side chain, isoleucine. The Ala has a smaller side chain than the Phe that it replaces in the substrate. The smaller side chain fits better into the smaller S1 subsite in the mutant enzyme.
In a second experiment, residues in subsites S1, S2, and S4, were changed to aspartate, either individually or in combinations. The mutants that were made are:
Mutant 1: Gly166 to Asp
Mutant 2: Gly166 to Asp and Asn62 to Asp
Mutant 3: Gly166 to Asp, Asn62 to Asp, and Tyr104 to Asp
The catalytic activity of the mutant enzymes was tested with the following peptide-p-nitroanilide substrates:
I Ala-Ala-Pro-Phe-p-nitroanilide
V Ala-Ala-Lys-Phe-p-nitroanilide
VI Arg-Ala-Lys-Arg-p-nitroanilide
VII Arg-Gly-Lys-Glu-p-nitroanilide
VIII Ala-Ala-Pro-Arg-p-nitroanilide
IX Ala-Gly-Glu-Arg-p-nitroanilide
X Phe-Gly-Lys-Arg-p-nitroanilide
XI Leu-Gly-Phe-Arg-p-nitroanilide
XII Ala-Ala-Lys-Arg-p-nitroanilide
XIII Arg-Gly-Ala-Arg-p-nitroanilide
XIV Arg-Gly-Lys-Phe-p-nitroanilide
Which substrate would be hydrolyzed most rapidly by each mutant enzyme?
Model Answer
Introduction of Asp residues into the subsites makes the mutant enzymes specific for substrates that have positively charged groups at the corresponding positions, but with other peptide residues unchanged from peptide I. The best substrates for the mutants would be:
Mutant 1: VIII Ala-Ala-Pro-Arg-p-nitroanilide
Mutant 2: XII Ala-Ala-Lys-Arg-p-nitroanilide
Mutant 3: VI Arg-Ala-Lys-Arg-p-nitroanilide