Biochimica etBiophysicaActa 828 (1985) 39-42
39
Elsevier BBA32131
T h e refolding of urea-denatured ribonuclease A is catalyzed by p e p t i d y l - p r o l y l
cis-trans isomerase
Gunter Fischer and Holger Bang Martin-Luther-Unioersit~t Halle, WB Biochemie (Abteilungff~r Enzymologie), Domplatz 1, 402 Halle (G.D.R.)
(ReceivedAugust 3rd, 1984)
Key words: Proteinrefolding;Prolineisomerization;cis-trans Isomerase
The refolding of urea-denatured ribonuclease A was measured at 0.31-3.1 mol. ! - i urea in the presence of various concentrations of peptidyl-prolyl cis.trans isomerase isolated from pig kidney. The rate of the slow CT-phase in the refoiding reaction was found to be sensitive to this enzyme. A rate enhancement proportional to the isomerase activity has been observed. The activity of the enzyme was assayed with Glt-Ala-Ala-Pro-Phe-4-nitroanilide. The catalytic activity of the isomerase against unfolded ribonuclease is suppressed after preincuhation of the enzyme with 0.001 mol. i-1 Cu2+, 0.01 mol. I - l H + and by heat inactivation. The results indicate the involvement of the c/s/tmns interconversion of proline peptide bonds during the refolding of ribonuclease A.
Introduction Various interpretations of the fast and slow phases in refolding of ribonuclease A have been proposed from a number of experiments [1-4]. The evidence is now rather strong that at least one of the slow phases in the refolding process exhibits the isomerization of the incorrect trans proline-93 to the cis isomer [5]. On the other hand, an enzyme has been recently found that is able to catalyze the c i s / t r a n s interconversion of proline-containing peptides [6]. This enzyme was isolated from pig kidney but is widespread in biological materials. The purpose of this study was to investigate the influence of this conformationally active enzyme on the various kinetic phases observed during the refolding of ribonuclease A. Chemical catalysis by very high concentrations of perchloric acid has been used to detect c i s / t r a n s participation in the formation of slow-folding species in the unfolded protein [8]. Enzyme catalysis in the refolding pathways, of course, would be the more native condition under which to check
the involvement of proline isomerization in protein folding. Secondly, it is important to know whether the peptidyl-prolyl cis-trans isomerase dan accommodate a protein chain as a substrate. Native ribonuclease A contains four proline residues in different states of conformation. They are mostly located near the C-terminus of the protein [9,10] and should be principally susceptible to the isomerase.
Materials and Methods Materials
Ribonuclease A was purchased from Ferak (Puriss., 45 U/mg). Pig kidney peptidyl-prolyl cis-trans isomerase was purified and assayed as described previously [6]. The assay is based on the trans specificity of bovine chymotrypsin for substrates with proline in the P2-position of the peptide chain [71. Instead of the DEAE-A50 ion-exchange chromatography in the purification procedure, a final
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40 run on an affinity column was applied. The affinity gel (AH-Sepharose 4B, Pharmacia) contained covalently bound peptide residues of the final structure . . . CH2-NH-OC-(CH2)3 -CONH-AIaAIa-Pro-PheNH-C6H4-(4-NO2). Activity was eluted with 0.005 mol. 1-1 Tricine buffer (pH 8.0). The enzyme preparation used in this work showed a specific activity [6] of 210 U / m g protein as assayed with 7.3.10 -6 mol. 1-1 cis-glutaryl-AlaAla-Pro-PheNH-CrH4-(4-NO2) at pH 7.8. At different urea concentrations the following isomerase activities against the same chromgenic peptide were observed in the final refolding buffer at pH 5.2: 0.31 mol. 1-1 urea, 34 U / m g ; 1.0 mol-1-1 urea, 25 U / m g ; 2.0 mol. 1-1 urea, 23 U / m g ; 3.1 mol. 1-1 urea, 21 U / m g . The enzyme exhibits its full activity of 210 U / m g in 0.31 mol. 1-1 urea at pH 7.8. Methods A Perkin-Elmer 356 spectrometer was used to monitor the refolding of ribonuclease. Unfolding and refolding conditions were strictly similar to those of Lin and Brandts [11]. Manual mixing with dead-time of 8-12 s was performed in 1 cm cells. The isomerase (stored in 0.005 mol. 1-1 Tricine buffer at pH 7.8) was preequilibrated in 0.05 mol. 1-1 sodium acetate buffer (pH 5.6). Tricine up to 0.004 mol. 1-1 has no effect on amplitudes and rate constants of ribonuclease refolding. Peeling-off the exponentials was used to calculate the rate constants. The experimental procedure as described in Ref. 12 was used to check the catalytic activity of the refolded ribonuclease, except that uridine 2',3'monophosphate (Serva) served as the substrate. Results
In order to correlate the results of our studies with the isomer-specific proteolysis, we have adapted our measuring conditions to the work of Lin and Brandts [11]. The change in the tyrosine absorbance at 287 nm was used to monitor the refolding. Additionally, the recovery of the catalytic activity of ribonuclease was followed in the 0.31 mol.1-1 urea experiment. Generally, in the refolding assays without ad-
ded isomerase activities, the same kinetic pattern was found as could be previously demonstrated by Lin and Brandts. At a final urea concentration of 2.0 mol. 1-1 the slow phase (~xv) is characterized by a single-exponential event which accounts for 78% amplitude and a rate constant of 2.6-10 -3 s -1 (lit.: 80%; 3.3.10 -3 s -1 [11]). Values of 83% and 1.5.10 -3 s -1 (lit.: 80%; 2.0.10 -3 s -1 [11]) have been found at 3.1 mol. 1-1 final urea. No attempt was made to investigate the very fast process (rND). Under these high-urea refolding conditions, no significant influence of peptidyl-prolyl cis-trans isomerase on both, amplitude and rate could be observed. This holds true up to 2.0 U / m g isomerase activity within the refolding buffer. By referring back to the data of Lin and Brandts at 0.31 mol. 1-1 and 1.0 mol. 1-1 urea, the single-exponential kinetics split off and a rather slow phase (rCT) appears. The data at 1.0 mol-1-1 urea are ~'xv 18% amplitude and 2.0.10 -2 s - l ; ~CT 60% amplitude and 6.8.10 -3 s -1 (lit.: ~'xv 16% and 2.6.10 -2 s-l; ZCT 64% and 8.3.10 -2 s -1 [11]). A more pronounced separation of the phase occurs at 0.31 mol.1-1 urea: ~'xv 50% and 3.7. 10 -2 s-l; TCT 30% and 8.8.10 -3 s -1 (lit.: ~'xv 50% and 4.5.10 -2 s - l ; ~'CT 30% and 1.2.10 -3 s -1 [11]). As demonstrated in Fig. 1 there is a markedly increase in rate of the CT-process in the presence of the isomerase at 0.31 mol. 1-1 urea. Qualitatively, the same picture has been observed at 1.0 mol- 1-1 urea. No rate enhancement was observed in the presence of 0.69 m g / m l bovine serum albumin. In this way any unspecific effect of protein was ruled out. The catalytic effect of the isomerase was found to be suppressed under several inhibitory conditions. Cu 2+ (1 h preincubation of the enzyme amount used in run 3 of Fig. 1 with 0.001 mol- 1- 1 C u 2 +) inactivate the isomerase and give during the refolding experiment kcT 8.1.10 -3 s -1. Furthermore, the same suppression of catalysis has been found by both heat inactivation (30 min at 80°C) and acid denaturation (20 min preincubation at pH 2.0). The conditions of preincubation are well known to inactivate completely the catalytic power of peptidyl-prolyl cis-trans isomerase against small peptide substrates [6].
41 Refolding experiments were also carried out at pH 7.8 because of the 6.2-times increased activity of the isomerase on small peptides. The rate enhancement for refolding of ribonuclease differ only to a small extent at pH 5.2 and pH 7.8. Further independent proof for catalysis of refolding could be attained by measurements of recovery of ribonuclease A activity. The data parallel the resuits found by the tyrosine absorbance measurements. They are included in Fig. 2. Keeping in mind the close rate constants of XY- and CT-phase, the peeling off must fail at higher concentrations of the catalyst. Therefore, the rate enhancements given in Fig. 2 are limited by the mechanism of refolding [5] and uncertainties of the numerical analysis. At high concentrations of the isomerase apparently the whole amplitude of the CT-phase disappears and only the uncatalyzable XY-phase is observed.
60 50
40
3C c u 2C c B 15 ~ , , ~X~x~X.~x.~ x
5- lc
0 0
\O'~l~ 0
Discussion
i@o Retolding time (s)
Fig. 1. Observed rate-time curves (measured at 287 nm) for the refolding of ribonuclease A without added peptidyl-prolyl cistrans isomerase (x), in presence of 0.24 U/ml isomerase(e)
and 0.6 U/ml isomerase(©). The conditionsare: 10.5°C,0.31 mol.l-1 urea in 0.05 mob 1-1 acetatebuffer (pH 5.2).
2.~ 20
•
X
1.5 o
1.0
0
0.5 110 210 Enzyme activity (u/ml) Fig. 2. Dependence of the rate constant kCT of refolding on the activity of the isomerase at 0.31 mol.1-1 urea (o) and 1.0 tool.1-1 urea (O). Recovery of ribonuclease activity at 0.31 mol.l - l (×).
Studying the refolding kinetics of urea denatured ribonuclease A in the presence of peptidylprolyl cis-trans isomerase from pig kidney, a distinct rate enhancement of refolding has been found. Only the CT-phase is open to the influence. As must be expected for an enzyme catalysis, the acceleration is suppressed by preincubation of the enzyme under inactivation conditions. Proteins themselves do not catalyze the refolding, as could be demonstrated by bovine serum albumin. Not many data about the specificity of the isomerase are available, but the direct involvement of proline isomerization in the refolding process of ribonuclease must now constitute proof of this. Since the proline peptide bond is accessible to the isomerase, one can assume the location of this residue (proline-93) on the surface in reversible unfolded ribonuclease. The involvement of proline-114 in the XY-phase (also cis in the native enzyme) is not supported by our results. On the other hand, it must be noted that peptidyl-prolyl cis-trans isomerase works on protein substrates. In comparison to Glt-Ala-AlaPro-PheNH-CrH4-(4-NO2), unfolded ribonuclease is apparently a poor substrate (approx. 103-fold activity of the tetrapeptide). But only after looking at the complete V/S-characteristic of catalysis is the discussion valid.
42 N o t h i n g is k n o w n a b o u t the physiological role in the isomerase. A l t h o u g h speculative, these results emphasize that c o n f o r m a t i o n i n t e r c o n v e r t i n g enzymes m a y be involved in the i n t e r m e d i a t e or final processing of the r i b o s o m a l released p e p t i d e chain. F u r t h e r m o r e , activation a n d d e a c t i v a t i o n of p r o l i n e - c o n t a i n i n g p e p t i d e h o r m o n e s b y limited proteolysis will give long-lived transients of unstab l e ratios of isomers [13]. Isomerases could avoid the slow d e c a y characteristic for the u n c a t a l y z e d cis / trans interconversion.
References 1 Chelis, C. and Yon, J. (1982) Protein Folding, pp. 362-373, Academic Press, New York 2 Kim, S.P. and Baldwin, R.L. (1982) Annu. Rev. Biochem. 51,459-489 3 Schmid, F.X. (1982) Eur. J. Biochem. 128, 77-80
4 Krebs, H., Schmid, F.X. and Jaenicke, R. (1983) J. Mol. Biol. 169, 619-635 5 Lin, L.-N. and Brandts, J.F. (1983) Biochemistry 22, 573-580 6 Fischer, G., Bang, H. and Mech, C. (1984) Biomed. Biochim. Acta 43, 1101-1111 7 Fischer, G., Bang, H., Berger, E. and Schellenberger, A. (1984) Biochim. Biophys. Acta 791, 87-97 8 Schmid, F.X. and Baldwin, R.L. (1978) Proc. FEBS Meet. 52, 173-185 9 Richards, F.M. and Wyckoff, H.W. (1971) in The Enzymes, 3rd Edn. (Bergmeyer, H.O., ed.), Vol. 4, pp. 467-470 10 Wlodawer, A., Bott, R. and Sj/51in, L.C. (1982) J. Biol. Chem. 257, 1325-1332 11 Lin, L.-N. and Brandts, J.F. (1983) Biochemistry 22, 564-573 12 Lin, L.-N. and Brandts, J.F. (1983) Biochemistry 22, 559-563 13 Fischer, G., Heins, J. and Barth, A. (1983) Biochim. Biophys. Acta 742, 452-462