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  • Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site.

Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site.

Biochemistry (1988-06-28)
J Pohl, B M Dunn
ABSTRACT

The possibility that pig pepsin has a cation binding specificity in its secondary binding subsites has been examined by the pepsin-catalyzed hydrolysis of a series of synthetic octa- to undecapeptide substrates. These chromophoric substrates are cleaved by pepsin in the phenylalanyl-p-nitrophenylalanyl (Phe-Nph) bond. Lys and Arg residues were placed into seven different positions in the substrates, and their effect on kcat and Km was examined between pH 2.8 and pH 5.8 (I = 0.1 M, 37 degrees C). Kinetic evidence indicates the existence in the enzyme binding subsites S4, S3, S2, S3', S4', and S5' of a group(s) which become(s) negatively charged at higher pH. For most substrates, the magnitude as well as the pH dependence of kcat was unaffected by the presence of Lys or Arg in these peptides. In contrast, changes up to 5 orders of magnitude were observed for Km, depending on the number of basic residues and on their positions in the sequence. Km for a group of substrates at pH greater than 5.5 was lower than 50 nM. Values for kcat/Km for some substrates exceed the level of 10(8) M-1 s-1. Therefore, the free energy derived from ionic interactions in secondary binding sites influences mostly the binding step on the reaction pathway. This result is in contrast to the previous observations that the length and the hydrophobic character of the substrate residues in some positions influence kcat with little effect on Km toward shorter substrates of pepsin [Fruton, J. (1976) Adv. Enzymol. Relat. Areas Mol. Biol. 44, 1-36].

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Pepsin−Agarose from porcine gastric mucosa, lyophilized powder, ≥30 units/mg dry solid
Sigma-Aldrich
Pepsin from porcine gastric mucosa, powder, ≥250 units/mg solid
Sigma-Aldrich
Pepsin from porcine gastric mucosa, powder, ≥400 units/mg protein
Sigma-Aldrich
Pepsin from porcine gastric mucosa, lyophilized powder, ≥2,500 units/mg protein (E1%/280)
Sigma-Aldrich
Pepsin from porcine gastric mucosa, lyophilized powder, ≥3,200 units/mg protein