Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1987 Aug 21;237(4817):909-13.
doi: 10.1126/science.3303334.

The catalytic role of the active site aspartic acid in serine proteases

C S Craik, S Roczniak, C Largman, W J Rutter

The catalytic role of the active site aspartic acid in serine proteases

C S Craik et al. Science. .

Abstract

The role of the aspartic acid residue in the serine protease catalytic triad Asp, His, and Ser has been tested by replacing Asp102 of trypsin with Asn by site-directed mutagenesis. The naturally occurring and mutant enzymes were produced in a heterologous expression system, purified to homogeneity, and characterized. At neutral pH the mutant enzyme activity with an ester substrate and with the Ser195-specific reagent diisopropylfluorophosphate is approximately 10(4) times less than that of the unmodified enzyme. In contrast to the dramatic loss in reactivity of Ser195, the mutant trypsin reacts with the His57-specific reagent, tosyl-L-lysine chloromethylketone, only five times less efficiently than the unmodified enzyme. Thus, the ability of His57 to react with this affinity label is not severely compromised. The catalytic activity of the mutant enzyme increases with increasing pH so that at pH 10.2 the kcat is 6 percent that of trypsin. Kinetic analysis of this novel activity suggests this is due in part to participation of either a titratable base or of hydroxide ion in the catalytic mechanism. By demonstrating the importance of the aspartate residue in catalysis, especially at physiological pH, these experiments provide a rationalization for the evolutionary conservation of the catalytic triad.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

Publication types

LinkOut - more resources