ELSEVIER Biochimica et Biophysica Acta 1249 (1995) 86-90

BB Biochi ~mic~a et Biophysica A~ta

The inhibition of a leaf proteinase by L-lysine homopolymers

Cristina A m a t o b Lucia Vaccari a , Ettore Balestreri a, * R o m a n o Fel ic io l i b

a lstituto di Biofisica, CNR, via S. Lorenzo 26, 56100 Pisa, Italy b Dipartimento di Fisiologia e Biochimica, Universith, Pisa, Italy

Received 24 May 1994; revised 5 August 1994; accepted 20 December 1994

Abstract

The role of interlinked positively charged amino acids in the mechanism of inhibition of a monomeric trypsin-like proteinase has been investigated using high molecular mass L-lysine homopolymers ranging from 3.8 to 109 kDa. The data show that the degree of polymerization enhances the inhibitory efficiency which is maximal for homopolymers with more than eighteen interlinked lysine residues. The inhibition is cooperative and, under the maximal inhibition conditions, nine lysine residues of the polymer are involved in the electrostatic binding to the enzyme. A limited conformational change of the protein molecule accompanies the transition from a fully active to a fully inactivated enzyme.

Keywords: Leaf proteinase; Cooperative inhibition; L-Lysine homopolymer

1. Introduct ion

Length-variable stretches of positively charged amino acid residues are frequently present in many proteins with a large variety of physiological functions. Regions rich in positively charged groups are reported in rheumatic dis- ease associated autoantigens [1,2], in regulatory and in cell surface proteins and in G protein-coupled receptors [3]. Furthermore cationic peptides are involved in phosphoryla- tion of calmodulin and other proteins [4-6] and in the calmodulin-stimulated cyclic nucleotide phosphodiesterase [7].

Evidence derived from experiments in which proteolytic enzymes were tested with a variety of naturally occurring or synthetic polycationic peptides indicates that cationic groups are involved in the proteinase-substrate or pro- teinase-inhibitor recognition processes [8]. Synthetic L- lysine polymers have been largely used as model sub- strates in numerous studies [9-11 ] directed to elucidate the mechanism of action of trypsin-like enzymes of both mam- malian and bacterial origin, since it is generally accepted that the positively charged side chains may mimic the trypsin site of attack. Furthermore, polycationic peptides are activators of the human erythrocyte multicatalytic pro-

* Corresponding author. Fax: + 39 50 553501.

Elsevier Science B.V. S S D 1 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 0 6 9 - 0

teinase [12] and numerous cationic compounds of different nature are powerful competitive inhibitors of plant pro- teinases [ 13-17].

Most of the reported experimental observations are ionic strength dependent and indicate the electrostatic na- ture of the primary event in the recognition and in the complex formation processes. But it is at present question- able if a general mechanism exist by which these com- plexes can evolve in so many different physiological re- sponses [ 18-20].

We have recently found [17,20] that a trypsin-like al- falfa leaf proteinase is inhibited by a large number of naturally occurring and synthetic polycationic compounds and that, depending on the number of the interlinked positive charges, the inhibition shifts from a pure competi- tive type, not sensitive to ionic strength, to a mixed competitive/non competitive type which is accompanied by a conformational change of the enzyme molecule; in this case both the inhibition and the conformational change are ionic strength dependent.

This observation prompted us to extend the study on the proteinase inhibition by a series of synthetic L-lysine ho- mopolymers with a high degree of polymerization. The final aim of this work is to investigate the mechanism by which a physiological response can be modulated by the primary electrostatic interaction of a protein with a polyca- tionic peptide.

C Amato et aL/ Biochimica et Biophysica Acta 1249 (1995) 86-90 87

2. Materials and methods

2.1. Materials

trations have been calculated from the fitted curves assum- ing the 98% as the maximum inhibition.

The alfalfa (Medicago sativa) leaf proteinase was puri- fied according to Tozzi et al. [21]. The monomeric enzyme has a molecular mass of 6,8 kDa and a p l of 4.5. Substrate and inhibitor specificity indicates that it is a trypsin-like proteinase.

DL-BAPNA ( N-a-benzoyl-DL-arginine-p-nitroanilide) and synthetic L-lysine homopolymers were purchased from Sigma (St. Louis, MO). Sigma guaranteed that the purity and the average molecular mass of each homopolymer have been checked by the A. Yaron and A. Berger method [22]. All other chemicals were of the highest quality commercially available.

The synthetic homopolymers of L-lysine were not, un- der any experimental condition, hydrolyzed when incu- bated in the presence of leaf proteinase as proven by amino acid analysis (data not shown).

2.2. Methods

Proteinase assay The proteinase activity was assayed by the spectropho-

tometric method of Erlanger et al. [23] using 3 - 10 -8 M purified proteinase and 0.2. 10 -3 M BAPNA, with poly(L-lysine) of various degrees of polymerization, at the molar lysine residue concentrations and at the ionic strength reported in the figures.

3. Results

Inhibition by L-lysine homopolymers The dependence of proteinase inhibition on the degree

of polymerization has been investigated using L-lysine homopolymers. Fig. 1 reports the inhibition of the pro- teinase activity as a function of L-lysine residue concentra- tion for the 3.8 kDa, 14 kDa and 27 kDa polymers. Analogous curves were obtained with 8.4 kDa, 52 kDa and 109 kDa which, for clearness, are not reported in the figure. The sigmoidicity of the curves clearly suggests a cooperative type of inhibition, and the data show that the inhibitory power increases with the degree of polymeriza- tion, with 150 of 1.08 IzM for the shortest polymer and 0.19 /zM for the longest one. The inset of Fig. 1 reports the Hill plots from which Hill coefficients ranging between 2.3 and 2.9 have been calculated. This suggests that more than one lysine residue of the homopolymer interacts with more than one site of the same enzyme molecule and indicates a rather complex inhibition mechanism.

As in the case of natural inhibitors polyamines and, partially, of the pentamer, the long-chain polypeptide inhi- bition is dependent on ionic strength. Fig. 2 reports the inhibition curves obtained with the 52 kDa poly(L-lysine) at different ionic strength ranging from 6 . 10 -3 /~ to 60" 10 -3 /z. The Hill coefficients have been calculated from the inset of Fig. 2; they are very similar to those

CD measurements Circular dichroism spectra were recorded at room tem-

perature on a Jasco J / 5 0 0 A spectropolarimeter using 3- 10 -7 M purified protei~Lase and the reported inhibitor lysine residue concentrations, in 20 mM Tris-HC1 buffer (pH 8.00). The data are reported as molar ellipticity ([ O ]).

Ionic strength The ionic strength (tL) was calculated from molar

concentration of the buffer and the conductivity was mea- sured with an ORION Microprocessor Conductivity Meter.

Data treatment Presented values are the means of three independent

determinations using different enzyme preparations and are statistically analyzed for standard error of the mean (S.E.M.).

The data of inhibition experiments fit well (correlation coefficients ranging from 0.98 to 0.99) with the Hill equation for cooperative kinetics v = Vo[I ]~/ (K+[I] ") where n is the Hill coefficiient and K is [I] ~ at v = 1 / 2 v o. We assume that the inhibition is 100% when the fitted curves fall short of the asymptote at a value inferior to the experimental S.E.M. The homopolymer saturating concen-

"~ 50 "-"

i~ Log [Lys]~M OI I I I !

0.0 0.5 1.0 1.5 2.0 2.5

[Lys'] p.M

Fig. 1. Inhibition of leaf proteinase by homopoly(L-lysine). The leaf protease was assayed in the presence of the reported concentrations of lysine residue as 3.8 kDa (v); 14 kDa (v); 27 kDa (D) homopolymers. The assays were carried out as reported under Section 2 in 20 mM Tris-HC1 buffer, pH 8.0. The inset shows the Hill plots for each ho- mopolymer.

88 C Amato et al. / Biochimica et Biophysica Acta 1249 (1995) 86-90

lOO

90

8o

70

60 2

~ 5o !=1 ,o ; o 3 0

1

0 1 2 3 4 5 6 7 8 9

[Lys]/zM

Fig. 2. Ionic strength dependence of the inhibition of the leaf proteinase by 52 kDa homopoly(L-lysine). The assays were carried out in Tris-HC1 buffer, pH 8.0 at 6 . 1 0 -3 p, ( Q ) ; 30.10 -3 I z (v); 6 0 . 1 0 -3 /z ( v ) . In the inset the Hill plots of the three curves are reported.

reports the 100% inhibition data for the long-chain ho- mopolymers, and, for comparison purposes, also those previously collected for the oligomers. They show that these ratio values vary of a factor of 104 from 6 .6 .105 for the dimer to 9 for the > 40-mer. This indicates that in the case of the homopolymers with more than 18 residues each enzyme molecule occupies a nine lysine residue-long seg- ment of the synthetic homopolymer so that a 109 kDa poly(L-lysine) could contemporarily bind 57 proteinase molecules. As a consequence also the molar ratio in- hibi tor /enzyme varies from 3.3 • l0 s to 1.5 - 10 -2 for the dimer and the 109 kDa polymer, respectively, as shown in Table 1.

The data of Table 1 and the sigmoidicity of the inhibi- tion curves strongly indicate a cooperative mechanism of inhibition supported by multiple electrostatic bonds and suggest that the enzyme inhibition could be achieved through a conformational change of the enzyme molecule, as already reported [17] for the pentalysine and for the naturally occurring inhibitors polyamines.

observed at low ionic strength and are unaffected by increasing salt concentrations. These results indicate that, independent of the extent of inhibition, the complex inhibi- tion mechanism does not vary with the salt concentration; furthermore, the dependence of the 150 on the ionic strength indicates that such mechanism is based on the electrostatic interactions.

Stoichiometry of the homopolymer / enzyme interaction When short length oligomers of L-lysine were used as

the leaf proteinase competitive inhibitors [20] a very high concentration of positively charged primary amino groups was necessary to obtain a 100% inhibition. In the course of the present work we observed that to a linear increase of the degree of polymerization corresponds an exponential increase of the inhibitory efficiency and that consequently the ratio [ENH~] / [enzyme] rapidly decreases. Table 1

Table 1 Stoichiometry of the homopolymer/proteinase interaction

Polymer [ •NH ~ ]a [inhibitor] a (L-lysine [Enzyme] [Enzyme] residues)

2 b 6.6.105 3.3.105 3 b 3.3.104 1.1 • 104

5 b 1.6" 104 3.3" 103 18 6.4.10 3.5 40 9.0 1.3 66 9.0 1.3.10- I 247 9.0 3.3.10 -2 519 9.0 1.5.10 -2

a The reported data refer to the 100% inhibition conditions as defined under Section 2. b The data are taken from Amato et al. [20].

Conformational changes induced by L-lysine homopoly- mers

We carried out CD measurements of the poly(L-lysine) and of the alfalfa leaf proteinase under a variety of experi- mental conditions. The band at about 220 nm originates from the contribution of a helix and /3 structure confor- mations, so its intensity gives an indication of the sec- ondary structure content of a protein in solution. Since poly(L-lysine) does not exhibit any CD signal in the 220 nm region at the concentration, pH and ionic strength conditions used, while the proteinase shows a well-defined negative band centered at 220 nm, we assume that any decrease observed in the 220 nm CD signal of the mixtures is only attributable to a loss of the secondary structure of the enzyme. The CD spectra of the proteinase in the wavelength range 210-250 nm are reported in Fig. 3: they refer to the proteinase alone and in the presence of poly(L- lysine) at various lysine residue concentrations. The data show that at low ionic strength ( 1 2 . 1 0 -3 /1,) the molar ellipticity decreases with increasing concentrations of the inhibitor cationic groups and that the induced conforma- tional change is prevented by high ionic strength. The inset of Fig. 3 also shows that the variation of the molar ellipticity does not linearly depend on the cationic group concentration: after a short lag it rapidly reaches the maximum loss in the range 1-3 /zM lysine residue con- centration. Further increase of the lysine concentration causes minor decrease in the secondary structure content. From the data of the inset of Fig. 3, a Hill coefficient higher than 1 has been calculated.

Relationship of inhibition to conformational change The interlinked positive charges of homopoly(L-lysine)

induce both a cooperative inhibition and a structural change

C. Amato et a l . / Biochimica et Biophysica Acta 1249 (1995) 86-90 89

0

i

fl 5 1 i J L I !

200 210 '?.20 230 240 250 260

;. (= )

Fig. 3. CD spectra of proteinase in the presence of 52 kDa homopoly(L- lysine). CD spectra in the absence (1) and in the presence of 0.6 /.L (2), 1.2 /z (3), 2.0 /z (4), 2.8 /.~ (5), 3.6 /x (6) lysine residue in 20 mM Tris-HC1 buffer (pH 8.0) at 6-10 -3 p. ( - - ) ; CD spectrum in the presence of 3.6 ~M lysine residue in Tris-HCl buffer (pH 8) at 120.10 -3 /z ( - - -) . The measurements were caJa-ied out as reported under Section 2. The inset shows the dependence of the molar ellipticity on the lysine residue concentration.

in the leaf proteinase molecule. Both features are sup- ported by electrostatic interactions as shown by their sensi- tivity to high ionic strength and are dependent on the degree of polymerization.

By using the 52 kDa homopolymer and low ionic strength conditions we measured the dependence of both features on the molar ral:io charged groups/enzyme. The

100

::

20A,~ , I I I !

0 2 4 6 8 10

30

2 0

10

0 12

N v

i: ¢

g

g 3

data are reported in Fig. 4: they show that the two curves are almost superimposable and reach their maximum at an [eNH~-]/[Enzyme] ratio range between 9 and 11, where the enzyme is fully inactivated and about 70% of the secondary structure content is still preserved.

4. Discussion

In a previous work [20], we reported that the inhibition of the alfalfa leaf proteinase by L-lysine oligomers shifts from a pure competitive type (dimer and trimer) to a mixed competitive/non competitive type (pentamer).

In the present paper the investigation on the dependence of the mode of inhibition on the degree of polymerization is extended to long-chain homopolymers, which proved to be powerful inhibitors. Furthermore they induce a confor- mational change of the enzyme molecule which in their presence undergoes a limited loss of the secondary struc- ture content.

For both the functional and structural features the pri- mary interactive event is supported by electrostatic interac- tions, the effect of which is not produced by the concentra- tion of the protonated amino groups per se but by inter- linked positive charges.

The inhibition and the conformational change are at their maximum when the enzyme molecule occupies a 9-11 residue-long segment of the synthetic polymer and more than one lysine residue of that segment contemporar- ily bind to the protein molecule. This evidence strongly suggests that the inhibition and the conformational change are strictly related.

The sigmoidicity of the inhibition curves, taken together with the Hill coefficients higher than 1 and the occurrence of a limited conformational change, are peculiar character- istics of the classical model of co-operatively regulated enzymes. We can therefore conclude that the inhibition of the alfalfa leaf monomeric proteinase by L-lysine 18-mer and higher polymers is a cooperative inhibition according to the Cornish-Bowden and Chrdenas [24] model. This cooperative molecular response of the leaf proteinase to the interlinked positive charges may contribute to explain the multiple observations so far described [1-7,12] on the modulation of biological activities by basic molecules through complex mechanisms not yet completely clarified. Furthermore the results described in this work may explain the sensitivity of the leaf proteolysis inhibition by polyca- tions (largely represented in nature) to local variation of ionic strength.

[~lH3 + ] / C q

Fig. 4. Dependence of the inhibition and of the conformational change on the [eNH~-]/[Enzyme]. The inhibition curve of homopoly(L-lysine) 52 kDa ( 0 ) as in Fig. 2; the percerLt of loss of [0]22 onm of the enzyme (O) is calculated from the data of Fig, 3.

Acknowledgements

This work has been financially supported by the Italian C.N.R., Programma finalizzato Chimica fine.

90 C. Amato et al. / Biochimica et Biophysica Acta 1249 (1995) 86-90

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