Lys40 but not Arg143 influences selectivity of angiotensin conversion by human α-chymase

Muilenburg, Diego J.; Raymond, Wilfred W.; Wolters, Paul J.; Caughey, George H.

doi:10.1016/s0167-4838(02)00224-8

Cited by 21 publications

(23 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Assay buffers contained phosphatebuffered saline with 0.01% Triton X-100 and 0.05% dimethyl sulfoxide, with concentrations of 4-NA substrates ranging from 71 to 425 M. Cleavage of angiotensin I was monitored by reverse phase high performance liquid chromatography of fragments generated by incubation with guinea pig and human chymases using modifications of approaches described previously (21,22). Peptidases were incubated with angiotensin I (8 -24 nM) in phosphate-buffered saline (pH 7.4) in aliquots of 50 l at 37°C.…”

Section: Expression and Mutagenesis Of Recombinant Chymases-formentioning

confidence: 99%

“…Cys 127 (chymotrypsinogen numbering) appears to be unique among serine peptidases; in the human and mouse enzyme, Phe is at this location, which is the principal site of autolytic cleavage in human chymase. Modeling results discussed in more detail below indicate that Cys 127 does not form an intramolecular disulfide bond; thus, guinea pig chymase has two unpaired cysteines, the other being Cys 22 , which is also unpaired in human chymase and presumably in other ␣-type chymases with a cysteine at this site. Guinea pig chymase contains two sites of predicted N-linked attachment to carbohydrates.…”

Section: Amino Acid Sequence Of Guinea Pig Preprochymase-as Shown Inmentioning

confidence: 99%

“…Even among chymotryptic chymases, there is a range of catalytic competence and target preference. For example, mammalian chymotryptic chymases vary in their tendency to hydrolyze after one or the other of two aromatic residues in angiotensin I (21,22). Cleavage at one site activates angiotensin I to angiotensin II, whereas cleavage at the other destroys activity.…”

mentioning

confidence: 99%

“…Cleavage at one site activates angiotensin I to angiotensin II, whereas cleavage at the other destroys activity. These preferences are influenced by sometimes subtle variations of amino acids in the vicinity of the substratebinding site (22). In the case of elastolytic chymases, notably the mouse chymase MCP-5 and its rat ortholog, the switch from chymotryptic to elastolytic specificity is attributable to natural mutation of a single amino acid (19,20).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Guinea Pig Chymase Is Leucine-specific

Caughey

Beauchamp

Schlatter

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the ␣ clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig ␣-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-ProLeu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe 8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by ␣ 1 -antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster ␣-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe-and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala 216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.

show abstract

Section: Expression and Mutagenesis Of Recombinant Chymases-formentioning

confidence: 99%

Section: Amino Acid Sequence Of Guinea Pig Preprochymase-as Shown Inmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Guinea Pig Chymase Is Leucine-specific

Caughey

Beauchamp

Schlatter

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…This side chain is aromatic for chymases and other chymotryptic enzymes. In the case of chymase-mediated hydrolysis of angiotensin I, interactions involving P1, P1Ј, and P2Ј are particularly important (24,25,27,28). However, for many serine peptidases (including chymase and cathepsin G hydrolyzing other peptide substrates), interactions on the other side of P1 (involving P4, P3, and P2) critically influence substrate binding and hydrolysis (29,30).…”

mentioning

confidence: 99%

Albumin Is a Substrate of Human Chymase

Raymond

Ruggles

Craik

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Human chymase is a chymotryptic serine peptidase stored and secreted by mast cells. Compared with other chymotryptic enzymes, such as cathepsin G and chymotrypsin, it is much more slowly inhibited by serum serpins. Although chymase hydrolyzes several peptides and proteins in vitro, its target repertoire is limited compared with chymotrypsin because of selective interactions in an extended substrate-binding site. The bestknown natural substrate, angiotensin I, is cleaved to generate vasoactive angiotensin II. Selectivity of angiotensin cleavage depends in major part on interactions involving substrate residues on the carboxyl-terminal (P1-P2) side of the cleaved bond. To identify new targets based on interactions with residues on the aminoterminal (P4 -P1) side of the site of hydrolysis, we profiled substrate preferences of recombinant human chymase using a combinatorial, fluorogenic peptide substrate library. Data base queries using the peptide (Arg-Glu-Thr-Tyr-X) generated from the most preferred amino acid at each subsite identify albumin as the sole, soluble, human extracellular protein containing this sequence. We validate the prediction that this site is chymase-susceptible by showing that chymase hydrolyzes albumin uniquely at the predicted location, with the resulting fragments remaining disulfide-linked. The site of hydrolysis is highly conserved in vertebrate albumins and is near predicted sites of metal cation binding, but nicking by chymase does not alter binding of Cu 2؉ or Zn 2؉ . A synthetic peptidic inhibitor, diphenyl N ␣ -benzoxycarbonyl-L-Arg-Glu-Thr-Phe P -phosphonate, was designed from the preferred P4 -P1 substrate sequence. This inhibitor is highly potent (IC 50 3.8 nM) and 2,700-and 1,300-fold selective for chymase over cathepsin G and chymotrypsin, respectively. In summary, these findings reveal albumin to be a substrate for chymase and identify a potentially useful new chymase inhibitor.

show abstract