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Active-site titration of horse urinary kallikrein.
Abstract: Horse urinary kallikrein was titrated with the reagent 4-nitrophenyl 4-guanidino-benzoate. The titration was shown to be dependent upon the concentration of the titrant. This finding, which distinguishes horse urinary kallikrein from other enzymes, is explained by the relatively small ratio between its rate of acylation and deacylation (k2/k3 = 16.8) and by a low affinity of the reagent (Km = 1.16 microM). By an appropriate kinetic treatment, it was possible to establish the relationship between the hydrolysis of 4-nitrophenyl 4-guanidinobenzoate and the actual concentration of the active enzyme.
Publication Date: 1984-03-01 PubMed ID: 6373553DOI: 10.1515/bchm2.1984.365.1.297Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research article is a scientific study on the titration of horse urinary kallikrein with a chemical reagent, focusing on the unique relationship between the concentration of the reagent and the enzyme’s distinctive properties.
Introduction
The research focuses on titration of a unique enzyme called horse urinary kallikrein using a chemical reagent known as 4-nitrophenyl 4-guanidino-benzoate. Titration is a common technique used in chemistry to determine the concentration of an unknown solution. In this case, horse urinary kallikrein was the unknown solution.
Methodology
- The titration of the horse urinary kallikrein was shown to be concentration-dependent, a trait that distinguishes it from other enzymes.
- The specific attributes that make horse urinary kallikrein unique were determined as the small ratio between its acylation and deacylation rates (k2/k3 = 16.8), and a low affinity of the reagent (Km = 1.16 microM).
- Acylation and deacylation rates refer to the speed at which chemical groups get added to, or removed from, a molecule, respectively. In this case, horse urinary kallikrein has a small acylation to deacylation ratio, indicating that chemical groups get removed from it more quickly than they are added.
- A low reagent affinity (Km) means that more substrate is required to achieve 50% enzyme saturation, indicating lower binding strength between the enzyme and reagent.
Results & Analysis
- Through a kinetic treatment, the researchers were able to establish a bridge between the hydrolysis of 4-nitrophenyl 4-guanidinobenzoate, a process by which water is induced to split a compound, and the actual concentration of the active enzyme. This provides insight into the unique kinetics of horse urinary kallikrein.
- Unlike other enzymes, such relationships and responses as designated by the study show that horse urinary kallikrein’s activity and characteristics come into play when titrated with the said reagent.
Conclusions
The researchers’ findings underscore the distinct traits of horse urinary kallikrein when titrated with 4-nitrophenyl 4-guanidino-benzoate. This unique relationship between the reagent’s concentration and the enzyme’s properties could have implications in developing protocols for using this enzyme in chemical reactions or in designing drugs and treatments.
Cite This Article
APA
Sampaio CA, Sampaio MU, Prado ES.
(1984).
Active-site titration of horse urinary kallikrein.
Hoppe Seylers Z Physiol Chem, 365(3), 297-302.
https://doi.org/10.1515/bchm2.1984.365.1.297 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Benzoates
- Binding Sites
- Cattle
- Endopeptidases / metabolism
- Horses
- Kallikreins / urine
- Kinetics
- Mathematics
- Pancreas / enzymology
- Protein Binding
- Serine Endopeptidases
- Substrate Specificity
- Swine
Citations
This article has been cited 10 times.- Lima AR, Juliano L, Juliano MA. Cyclic, linear, cycloretro-isomer, and cycloretro-inverso peptides derived from the C-terminal sequence of bradykinin as substrates or inhibitors of serine and cysteine proteases. Protein J 2004 May;23(4):287-94.
- Fogaça SE, Melo RL, Pimenta DC, Hosoi K, Juliano L, Juliano MA. Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins. Biochem J 2004 Jun 15;380(Pt 3):775-81.
- Nunes VA, Gozzo AJ, Sampaio MU, Juliano MA, Sampaio CA, Araujo MS. Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site. J Protein Chem 2003 Aug;22(6):533-41.
- Pimenta DC, Fogaça SE, Melo RL, Juliano L, Juliano MA. Specificity of S'1 and S'2 subsites of human tissue kallikrein using the reactive-centre loop of kallistatin: the importance of P'1 and P'2 positions in design of inhibitors. Biochem J 2003 May 1;371(Pt 3):1021-5.
- Pimenta DC, Chao J, Chao L, Juliano MA, Juliano L. Specificity of human tissue kallikrein towards substrates containing Phe-Phe pair of amino acids. Biochem J 1999 Apr 15;339 ( Pt 2)(Pt 2):473-9.
- Di Ciero L, Oliva ML, Torquato R, Köhler P, Weder JK, Camillo Novello J, Sampaio CA, Oliveira B, Marangoni S. The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds. J Protein Chem 1998 Nov;17(8):827-34.
- Pimenta DC, Juliano MA, Juliano L. Hydrolysis of somatostatin by human tissue kallikrein after the amino acid pair phe-Phe. Biochem J 1997 Oct 1;327 ( Pt 1)(Pt 1):27-30.
- Portaro FC, Cezari MH, Juliano MA, Juliano L, Walmsley AR, Prado ES. Design of kallidin-releasing tissue kallikrein inhibitors based on the specificities of the enzyme's binding subsites. Biochem J 1997 Apr 1;323 ( Pt 1)(Pt 1):167-71.
- Chagas JR, Portaro FC, Hirata IY, Almeida PC, Juliano MA, Juliano L, Prado ES. Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallikreins. Biochem J 1995 Feb 15;306 ( Pt 1)(Pt 1):63-9.
- Del Nery E, Chagas JR, Juliano MA, Prado ES, Juliano L. Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments. Biochem J 1995 Nov 15;312 ( Pt 1)(Pt 1):233-8.
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