Investigating the s-2 subsite selectivity of alkaline proteases in hydrolysis of diastereo-peptide esters and molecular-modeling interpretation.
Diastereomeric peptide-esters (Ala-Ala-AA2-Phe-OMe, AA2 = Gly, D- or L-Ala, Pro, Phe, Lys, and Glu) have been used as substrates, and the kinetic constants (Kcat and Km) of the three alkaline proteases, subtilisin Carlsberg, alcalase, and Nagarse (subtilisin BPN') catalyzed ester-hydrolysis, were measured to investigate the selectivity of the enzyme-catalyzed peptide esterhydrolysis. All three proteases preferred the substrate which had a small side-chain at the s-2 site. Thus, the substrates with a bulky side-chain at the p-2 site such as Phe, Pro, Glu, and Lys, hydrolyzed with a rate of about one-tenth that of Ala at the p-2 site, and the Kcat decreased as the size of the p-2 amino acid residue increased. The diastereoselectivity of the alkaline protease-catalyzed hydrolysis of each diastereomeric pair depended on the size of the amino acid residue at the p-2 position of the substrate. The substrates with a bulky side-chain at the p-2 site hydrolyzed with higher diastereoselectivity than did the substrates with a small side-chain at the p-2 site. Molecular modeling of the enzyme-substrate complex show that: for the enzyme complexed with a substrate which has L-L-L-L configuration, each residue of the L-L-L-L tetrapeptide filled in and was completely enclosed by the cleft of the four subsites of the enzyme. The side-chains of the residues were identically positioned within the pocket of the binding-site. For the complex of enzyme with substrate of L-L-D-L, the side-chain of the D-amino acid residue was far away from the s-2 subsite of the enzyme, and had close contact with the side-chains of Leu-126 and Ile-107 of the enzyme.
Chen
S T
ST
Institute of Biological Chemistry, Academia Sinica, Taiwan, R.O.C.
Tu
C C
CC
Chen
S Y
SY
Huang
H C
HC
Wang
K T
KT
D000595:Amino Acid Sequence; D001665:Binding Sites; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions; D013237:Stereoisomerism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*; D014867:Water
D009842:Oligopeptides; D012996:Solutions; D014867:Water; D013381:Subtilisins