Use of secondary isotope effects and varying pH to investigate the mode of binding of inhibitory amino aldehydes by leucine aminopeptidase

Andersson, Lars I.; MacNeela, John P.; Wolfenden, Richard

doi:10.1021/bi00323a014

Cited by 28 publications

(23 citation statements)

References 16 publications

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“…However the pH optima for aminopeptidase activity in control leaf extracts was found to be neutral (pH 7.0). Recent X-ray crystallographic studies have suggested that the alkaline pH optima may reflect the need for the substrate's N-terminus to be in an unprotonated state to facilitate interaction with the hydrophobic catalytic core of enzyme [26,27]. Interestingly our findings suggest that pigeonpea inducible aminopeptidase may have alkaline pH optima since pH optima for enzyme activity was alkaline in the leaf extracts of treated plants.…”

Section: Discussionmentioning

confidence: 52%

Induction of leucine aminopeptidase (LAP) like activity with wounding and methyl jasmonate in pigeonpea (Cajanas cajan) suggests the role of these enzymes in plant defense in leguminosae

Lomate

Hivrale

2011

Plant Physiology and Biochemistry

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Section: Discussionmentioning

confidence: 52%

Induction of leucine aminopeptidase (LAP) like activity with wounding and methyl jasmonate in pigeonpea (Cajanas cajan) suggests the role of these enzymes in plant defense in leguminosae

Lomate

Hivrale

2011

Plant Physiology and Biochemistry

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“…Further biochemical characterization of the porcine LAP, E. coli PepA and tomato LAP‐A showed that all three LAPs had high temperature and alkaline pH optima. Recent X‐ray crystallographic studies have suggested that the alkaline pH optima may reflect the need for the substrate’s N‐terminus to be in an unprotonated state to facilitate interaction with the hydrophobic catalytic core of LAP [13,51]. It is also possible that the tomato LAP‐A’s alkaline pH optimum may be related to its site of action within the plant cell or its role in plant defense.…”

Section: Discussionmentioning

confidence: 99%

Overexpression, purification and biochemical characterization of the wound‐induced leucine aminopeptidase of tomato

Holzer

Walling

1999

European Journal of Biochemistry

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Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A). While the expression of LapA genes are well characterized, the specificity of the LAP-A enzyme has not been studied. The LAP-A preprotein and mature polypeptide were overexpressed in Escherichia coli. PreLAP-A was not processed and was inactive accumulating in inclusion bodies. In contrast, 55-kDa mature LAP-A subunits assembled into an active, 357-kDa enzyme in E. coli. LAP-A from E. coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E. coli PepA) homologues. Similar to the porcine and E. coli enzymes, the tomato LAP-A had high temperature and pH optima. Mn 2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow-binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs. The substrate specificities of porcine, E. coli and tomato LAPs were determined using amino-acid-p-nitroanilide and -b-naphthylamide substrates. The tomato LAP-A preferentially hydrolyzed substrates with N-terminal Leu, Met and Arg residues. LAP-A had substantially lower levels of activity on other chromogenic substrates. Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted.

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“…Compounds containing the aldehyde component often serve as very effective inhibitors of proteolytic enzymes such as elastase (26), papain (27)(28)(29), chymotrypsin (30), amidase (31), asparaginase (32), leucine aminopeptidase (33,34), and the serine protease SGPA from Streptomyces griseus (35). The enhanced electrophilicity at the carbonyl carbon of the aldehyde invites a fourth saturating ligand, such as a water molecule, exemplified by aldehyde-hydrate equilibria in aqueous solution.…”

mentioning

confidence: 99%

Binding of a possible transition state analogue to the active site of carboxypeptidase A.

Christianson¹,

Lipscomb²

1985

Proc. Natl. Acad. Sci. U.S.A.

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The mode of binding of the competitive inhibitor 2-benzyl-3-formylpropanoic acid to the active site of carboxypeptidase A has been studied by x-ray diffraction methods to a resolution of 1.7 A. The actual species bound to the enzyme was determined to be the gem-diol resulting from covalent hydration at the aldehyde carbonyl. Details relating to the process of association of inhibitor with enzyme are unknown at this time: the free aldehyde could initially bind to the enzyme and subsequently undergo catalytic hydration; or, the hydrate itself could be the species initially binding to the enzyme, because it does exist to a high degree (25%) in aqueous solution. Nevertheless, the structure of the complex reported is reminiscent of a possible tetrahedral intermediate that would be encountered in a general base hydrolytic mechanism. Of course, other mechanistic proposals, such as the anhydride pathway, cannot be ruled out simply on the basis of the structure of this enzyme-inhibitor complex.The metalloenzyme carboxypeptidase A, (CPA; peptidyl-Lamino-acid hydrolase, EC 3.4.17.1) is an exopeptidase of molecular weight 34,472, containing one zinc ion bound to a single polypeptide chain of 307 amino acids (1-3). Its biological function is the hydrolysis of COOH-terminal amino acids from polypeptide substrates, and it exhibits preferred specificity toward substrates possessing large hydrophobic COOH-terminal residues. The hydrolytic mechanism of CPA has received considerable kinetic and structural study, and the enzyme has been the subject of many recent reviews (4-8). Important residues for substrate binding and catalysis are arginine-145, tyrosine-248, glutamate-270, and Zn(II); a catalytic role for arginine-127 is also considered in light of its interaction with a substrate in the x-ray structure of an apoenzyme complex (9). However, important details of the hydrolysis remain as yet unclear. Two probable mechanisms include the initial nucleophilic attack of the y-carboxylate of residue glutamate-270 at the scissile carbonyl carbon of the substrate with subsequent formation of an anhydride intermediate, or the attack of a water molecule at the scissile carbonyl promoted by glutamate-270 and/or Zn2+.Structural investigations of CPA have been aided in part by x-ray crystallographic studies on the native enzyme (10-12) and its complexes with different inhibitors (12-16). When considering the mechanistic implications of the three-dimensional structure of enzyme-inhibitor complexes, it is important to note the distinction between the use of substrate analogues, products or product analogues, and transitionstate analogues as inhibitors. Each of these classes of inhibitors can provide valuable information about the association of substrate with enzyme. However, if one supposes a correlation between inhibitor affinity and actual transitionstate resemblance, as presumed from considerations of enzyme-transition-state complementarity (17,18) configuration at the formerly carbonyl carbon gives rise to a structure that could...

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Use of secondary isotope effects and varying pH to investigate the mode of binding of inhibitory amino aldehydes by leucine aminopeptidase

Cited by 28 publications

References 16 publications

Induction of leucine aminopeptidase (LAP) like activity with wounding and methyl jasmonate in pigeonpea (Cajanas cajan) suggests the role of these enzymes in plant defense in leguminosae

Induction of leucine aminopeptidase (LAP) like activity with wounding and methyl jasmonate in pigeonpea (Cajanas cajan) suggests the role of these enzymes in plant defense in leguminosae

Overexpression, purification and biochemical characterization of the wound‐induced leucine aminopeptidase of tomato

Binding of a possible transition state analogue to the active site of carboxypeptidase A.

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