Abstract

Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2' (Leu or Asp), and P4' (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2' (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4' (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K(D) 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K(D) of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys(51), Arg(56), and Arg(80)) on the surface of human CTRC that interact with the P4' acidic residue of the inhibitor. The acidic preference of CTRC at P4' is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.

Highlights

  • Are produced as inactive proenzymes and their activation is spatially restricted to the duodenum and proceeds in a cascade-like manner

  • We demonstrated that human chymotrypsin C (CTRC)4 regulates activation and degradation of human cationic trypsinogen and trypsin [1, 2]

  • The results suggest that a unique electrostatic interaction between a cluster of basic residues on CTRC and the P4Ј acidic amino acid residue of CTRC substrates is an important determinant of CTRC specificity

Read more

Summary

CTRC cleavage sites

Activation of procarboxypeptidases is initiated by trypsin-mediated proteolytic cleavages at the C-terminal end of the propeptides. The CTRC-catalyzed regulatory functions are highly specific as other human pancreatic chymotrypsins and elastases exhibit no such activity [1, 2, 7]. This may be partly due to the primary (P1) specificity of CTRC, which is chymotrypsin-like, but CTRC exhibits higher activity on leucyl peptide bonds than chymotrypsin A or B (8 –10). Other determinants may be important, as suggested by the preponderance of acidic amino acid residues on the prime side of the CTRCspecific regulatory cleavage sites (Table 1). We fully randomized six protease binding loop positions in the chymotrypsin inhibitor SGPI-2, displayed the library on M13 phage, and selected tight binding variants against CTRC. The results suggest that a unique electrostatic interaction between a cluster of basic residues on CTRC and the P4Ј acidic amino acid residue of CTRC substrates is an important determinant of CTRC specificity

EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call