Transformation of active-site lysine in naturally occurring trypsin inhibitors.  A basis for a general mechanism for inhibition of proteolytic enzymes

Haynes, Royce; Feeney, Robert E.

doi:10.1021/bi00848a026

Cited by 62 publications

(18 citation statements)

References 14 publications

(25 reference statements)

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“…Similar proposals have been already presented for the interaction of trypsin with ovomucoid, lima bean and pancreatic secretory inhibitors [29,32]. Recent crystallographic data also indicate that structural alterations in or-chymotrypsin and the pancreatic trypsin inhibitor occur during association [33].…”

Section: Discussionsupporting

confidence: 73%

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

Vincent¹,

Lazdunski²

1973

European Journal of Biochemistry

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1. The association constant, Ka, of the 1 : i complex formed between a-chymotrypsin and the pancreatic trypsin inhibitor is 110 pM-l a t pH 8.0 and 25 "C. The second-order rate constant for the association, Ea, is 110 mM-l s-l and the first-order rate constant for the dissociation, E d , is lo-, s-l under the same conditions. Thermodynamic parameters for complex formation a t 25 "C, pH 8.0 are A GaO = -li .O kcal x mol-l, A Ha'' = 3 kcal x mol-l and A 8 , ' ' = 48 cal.-mol-l * K-l. 2.Temperature and pH-dependences of the rate constants k, and k d have been studied. 3. The difference in stability between the a-chymotrypsin * inhibitor complex and the trypsin -inhibitor complex (Ka = 16 pM-l, d Q$ = -18.1 kcal x mol-l) is due essentially to differences of ka values. The dissociation of the a-chymotrypsin inhibitor complex is i.5 x 104 times faster than that of the trypsin -inhibitor complex. Differences in Ka and ka values are discussed in terms of differences in stabilizing interactions.4. The Cys,,-Cys,, disulfide bridge of the inhibitor, which is highly succeptible to reduction when the inhibitor is free, is masked in the a-chymotrypsin -inhibitor complex. Reduction of the Cys,,-Cys,, bridge in the free inhibitor does not prevent association with a-chymotrypsin. Values of k , and Ed for the formation of the 1 : 1 a-chymotrypsin * reduced-inhibitor complex and for the complex formed with the native inhibitor are very similar. This situation differs from that observed with the trypsin * pancreatic-inhibitor complex. I n that case, reduction of Cys,,-Cys,, bridge considerably decreases the stability of the complex formed with trypsin ; K , is decreased by a factor of 3 x lo4 and kd is increased by a factor of 8.6 x los.I n spite of their importance in control systems, only a limited number of interactions between heterologous proteins or between peptides and proteins have been extensively studied. However it is well known that many such associations are very tight. Protein and peptide hormones such as insulin and the thyroid-stimulating hormone [4] for example act a t very low doses. Dissociation constants for the complexes they form with their receptors are lower that 10 nM.We reported previously the results obtained for the formation of the complex between trypsin and pancreatic trypsin inhibitor. This association is one of the security devices which avoid accidental activation of trypsinogen in the pancreas. The association between trypsin and the pancreatic trypsin inhibitor is unusually strong. The dissociation constant, Kd, of the 1 : 1 complex is 60 fM at pH 8.0,We report here the results obtained for the formation of the complex between ol-chymotrypsin and pancreatic trypsin inhibitor. Each of the partners in the complex is well characterized. Both their covalent and three-dimensional structures in the crystalline state are available [6-111 and a considerable amount of work has been devoted to the analysis of the components of their active sites. The essential catalytic groups of ol-chymotrypsin are ; the ...

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Section: Discussionsupporting

confidence: 73%

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

Vincent¹,

Lazdunski²

1973

European Journal of Biochemistry

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“…The requirement of either an arginine or a lysine residue in the active site of trypsin inhibitors is well established (Ozawa and Laskowski, 1966;Haynes and Feeney, 1968). To see which residue is responsible for trypsin inhibition, the arginine residue of inhibitor LA-1 and the lysine and arginine residues of inhibitor LA-2 were examined.…”

Section: Inhibition Sitesmentioning

confidence: 99%

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification, properties, and amino acid sequences

et al. 1996

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Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is at pH 4.55 for LA-1 and at pH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 A. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0 x 10(9) M-1 sec-1 for LA-1 and 0.8 x 10(9) M-1 sec-1 for LA-2 and that of K2HPO4 quenching is 1.6 x 10(11) M-1 sec-1 for LA-1 and 1.2 x 10(11) M-1 sec-1 for LA-2. Analysis of the circular dichroic spectra yields 40% alpha-helix and 60% beta-turn for La-1 and 45% alpha-helix and 55% beta-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzyme-inhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.

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“…Previous work (Hunter & Ludwig, 1962;Wofsy & Singer, 1963;Reynolds, 1968;Haynes & Feeney, 1968;Benisek & Richards, 1968) has indicated that the reaction of aliphatic and aromatic imidates with proteins is confined to amino groups. Determination of the extent of reaction of methyl iodopicolinimidate with oxidized insulin by difference spectroscopy and bv assaying the free amino-group content by reaction with trinitrobenzenesulphonic acid gave results that were in excellent agreement, indicating that the reaction of oxidized insulin with this reagent was also confined to the amino groups.…”

Section: Discussionmentioning

confidence: 99%

“…Appropriately substituted imidates satisfy these criteria. Imidates are able to react with monosubstituted amines to give the corresponding N-monosubstituted amidines (Roger & Neilson, 1961;Hand & Jencks, 1962) and, in their reaction with proteins, imidates have been shown to be completely specific for amino groups (Hunter & Ludwig, 1962;Wofsy & Singer, 1963;Reynolds, 1968;Haynes & Feeney, 1968;Perham & Richards, 1968). Further, the reaction of protein amino groups with imidates can be accomplished under mild conditions (pH 8-10) and the resulting monosubstituted amidines have pKL values in the range 11.5-12.5 (Schwarzenbach & Lutz, 1940;Albert et al, 1948), i.e.…”

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confidence: 99%

The reaction of protein amino groups with methyl 5-iodopyridine-2-carboximidate. A possible general method of preparing isomorphous heavy-atom derivatives of proteins

Riley

Perham

1973

Biochemical Journal

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1. The synthesis of methyl 5-iodopyridine-2-carboximidate and its reaction with amino groups of model compounds and performic acid-oxidized insulin are described. The reagent was designed to introduce heavy atoms into specific sites in proteins. 2. Specific reaction with the amino groups of oxidized insulin can be achieved under reasonably mild conditions giving rise to the corresponding N-monosubstituted amidines. 3. The extent of reaction of this reagent with protein amino groups can be readily determined by difference spectroscopy. Modification of lysine residues inhibits tryptic cleavage at such residues, and this can be of assistance in establishing the site of modification in the primary structure. 4. Evidence is presented to show that methyl 5-iodopyridine-2-carboximidate can react specifically, at pH5.0, with the aromatic amino group of 3-amino-l-tyrosine; the final product of this reaction is a 2-arylbenzoxazole. 5. The use of this reagent as a general method for preparing heavy-atom isomorphous derivatives of proteins is discussed.

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Transformation of active-site lysine in naturally occurring trypsin inhibitors. A basis for a general mechanism for inhibition of proteolytic enzymes

Cited by 62 publications

References 14 publications

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification, properties, and amino acid sequences

The reaction of protein amino groups with methyl 5-iodopyridine-2-carboximidate. A possible general method of preparing isomorphous heavy-atom derivatives of proteins

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