# ANTICHYMOTRYPSIN INTERACTION WITH SERINE PROTEASES

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 1993 · $198,865

## Abstract

Our work is directed toward obtaining a fundamental understanding of the
basis of serpin interaction with serine proteases.  The particular serpin
we have chosen to study in detail is alpha1-antichymotrypsin (ACT).  ACT is
a relatively specific inhibitory of chymotrypsin-like serine proteases
[chymotrypsin (Chtr), cathepsin G (CatG)].  In contrast, the serpin alpha1-
protease inhibitor (alpha1-PI) is relatively non-specific, being a potent
inhibitor of Chtr, trypsin, and elastase (Travis and Salvesen, 1983).  In
the experiments described below we seek to gain a full understanding of the
interaction of ACT with Chtr and CatG, and to test our understanding by
redesigning ACT, through site-specific mutagenesis, to change its
specificity.

First, we will carry out site-specific mutagenesis and chemical
modification in order to identify amino acid residues in ACT that are
important for its interaction with Chtr (CatG).  We will seek answers to
the following questions:  a) how much of the reactive loop of ACT is
covered on forming a complex with Chtr (CatG)/; b) what residues within the
reactive loop are most important for interaction with Chtr (CatG), and
precisely how do they affect the interaction?; c) how much of the protein
is required in order for it to act as an inhibitor?; d) are residues
outside the reactive loop important for interaction with Chtr (CatG)?

Second, we will make use of site-specific mutagenesis to introduce reporter
groups into the reactive loop, and employ stopped-flow spectrophotometry to
detect rapidly formed intermediates in the interaction of ACT (and ACT
variants) with Chtr (CatG) and determine rate constants for their
appearance and disappearance.

Third, we will identify which new sites of ACT are cleaved during the newly
described "locking" step in the interaction of ACT and Chtr and determine
whether suitable substitution at these sites can significantly alter the
mechanism of ACT action.

Fourth, we will use site-specific mutagenesis to ask whether by
substitution of the active loop of alpha1-PI for the active loop of ACT,
and suitable further variation within the loop but leaving the rest of the
ACT molecule unchanged, we can convert ACT into an effective inhibitor of
human neutrophil elastase (HNE).

Fifth, we will use calorimetric methods to the overall equilibrium
constants and heats of enthalpy for ACT and ACT variants with Chtr (CatG).

Sixth, we will determine the new N-termini produced on reaction of the
L358R variant of ACT with Chtr to address the question of the significance
of the site of cleavage within the reactive loop for antiprotease activity.

Seventh, we will attempt to grow crystals of ACT and of the ACT complex
with Chtr suitable for X-ray diffractional analysis at high resolution.
Such analysis will be carried out if such crystals are grown.

## Key facts

- **NIH application ID:** 3122508
- **Project number:** 5R01AG010599-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **SAM.gov UEI:** GM1XX56LEP58
- **Principal Investigator:** BARRY S. COOPERMAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1993
- **Award amount:** $198,865
- **Award type:** 5
- **Project period:** 1991-09-01 → 1996-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3122508

## Citation

> US National Institutes of Health, RePORTER application 3122508, ANTICHYMOTRYPSIN INTERACTION WITH SERINE PROTEASES (5R01AG010599-03). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3122508. Licensed CC0.

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