- Email: [email protected]
Autolysis and inhibition of proteinase K, a subtilisin-related serine proteinase isolated from the fungus Tritirachium album Limber
176
Biochimica et Biophysica Acta 954 (1988) 176-182
Elsevier BBA33129
Autolysis and inhibition of proteinase K, a subtilisin-related serine proteinase isolated from the fungus Tritirachium album Limber
Ji~rgen Bajorath, Wolfram Saenger and Gout Pada Pal * Institut ]'dr Kristallographie, Freie Universitiit Berlin, Berlin (F.R.G.)
(Received 13 October 1987) Key words: ProteinaseK; Serineproteinase; Protein-protein interaction; Autodigestion; Enzymeinhibition; Syntheticinhibitor; (T. album) The activity of proteinase K (EC 3.4.21.14), a subtilisin-related serine proteinase, was assayed with azoalbumin that showed non-expected behavior in substrate saturation curve because of interaction between albumin molecules. Succinyl-(Aia)n-p-nitroanilide with n = 2 and 3, yielded specific activities of 3.5, 13 u n i t s / r a g protein, respectively, reflecting a chain length dependence. The influence of peptide chain length on binding to proteinase K was also observed using mono- and dipeptide chloromethyi ketone inhibitors. They showed a maximum inhibition. They showed a maximum inhibition of proteinase K in solution of only about 50% even at a more than 20-fold molar excess. With the above substrates, the Vmax is not affected in presence of 10, 20 and 30% methanol, but the K m differs remarkably, suggesting competitive inhibition. The activity of proteinase K shows a maximum at 37°C, and a temperature profile with more than 80% maximum activity in the range 20-60 o C. Autolysis of the enzyme is observed during sample preparation for SDS-gel electrophoresis and at low concentration (0.01 m g / m l ) in aqueous solution. It does not o c c u r at higher proteinase K concentrations at or above 1.0 m g / m l , consistent with crystallographic studies. Introduction
Proteinase K (EC, 3.4.21.14) isolated from fungus, Tritirachium album Limber, is one of the most active serine proteinases. It can hydrolyse even native keratin and remains active in presence
Abbreviations: Suc-(Ala)3-pNA, succinylalanylalanylalanylpara-nitroanilide; Suc-(Ala)2-pNA, succinylalanylalanyl-para-
nitroanilide; Z-Ala-Phe-CK, carbobenzoxyalanylphenyl chloromethyl ketone; Z-Ala-Ala-CK, carbobenzoxyalanylalanyl chloromethylketone; Z-Phe-CK, carbobenzoxyphenyl chloromethylketone. * Present address: European Molecular Biology Laboratory, Heidelberg, F.R.G. Correspondence:J. Bajorath, Institut ftir KristaUographie,Freie Universit~it Berlin, TakustraBe6, D-1000, Berlin, F.R.G.
of sodium dodecyl sulfate and urea [1]. The enzyme shows an unusual retention in a colunm of Sephadex G-75 which led to an erroneous moleculax weight determination of 18500 [1]. After the elucidation of its three-dimensional structure, the molecular weight of proteinase K was calculated to 27000 [2] in good agreement with the value determined by SDS-polyacrylamide gel electrophoresis [2]. Recently, the true molecular weight has been evaluated as 28 930 from the amino-acid sequence [3]. Although the three-dimensional structure of proteinase K has been refined at 1.5 resolution by X-ray crystallographic methods [4] and is thus known in great detail, the biochemical properties of the enzyme are not yet well characterized. The present studies on the biochemical behavior of proteinase K should help to evaluate further the structure-function relationship of this enzyme.
0167-4838/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
177
Materials and Methods
Materials Proteinase K was purchased from E. Merck, Darmstadt, F.R.G.; azoalbumin from Sigma Chemicals; albumin from Boehringer, Mannheim; Suc-(Ala),-pNA from Serva Feinbiochemica, Heidelberg, F.R.G. The synthetic inhibitor, Z-AlaPhe-CK, was supplied by Dr. Burgert, Institut fiir Organische Chemie, Freie Universit~it Berlin, ZAla-Ala-CK and Z-Phe-CK were obtained from Prof. Jany, Universit~t Stuttgart. All other commonly available reagents were of analytical grade. For characterization of commercial proteinase K, standard polyacrylamide gel electrophoresis was carried out at pH 7.2 as described [5]. The material was pretreated with SDS and 2-mercaptoethanol. In absence of SDS, polyacrylamide gel electrophoresis was performed at pH 4.3. Buffers A and B were prepared according to Ebeling et al. [1]. Buffer A: 0.01 M Tris-HC1, 5 mM CaC12 (pH 8.0); and buffer B: 50 mM TrisHC1, 5 mM CaC12 (pH 8.0).
Purification of proteinase K A 10% aqueous solution of the commercial product was subjected to gel-filtration using an Sephadex G-75 column (2 x 70 cm) which was pre-equilibrated with buffer A at room temperature. The protein was eluted with the same buffer at a flow rate of approx. 40 m l / h and fractions of 6 ml were collected. The presence of protein was monitored by absorbance measurements at 280 nm and the proteinase K activity was assayed with azoalbumin as substrate. Active fractions were pooled, dialysed exhaustively against 1 m M calcium acetate at 4 ° C , and lyophilized. The material was stored at 4 ° C till further use.
Assay of enzyme activity The proteolytic activity of proteinase K was monitored using azoalbumin as substrate, and for the routine assay its amidase activity was followed using Suc-(Ala)3-pNA as substrate. Prior to the assay, lyophilized proteinase K was dissolved at a concentration of 1 m g / m i in buffer B and incubated routinely at 20 ° C for 10 min.
Substrate saturation curve For a data point on the substrate saturation
curve, experiments were performed with azoalbumin and Suc-(Ala)3-pNA as substrates. (a) Azoalbumin. 1 ml of the assay mixture containing 10 ~tg of the enzyme and a final substrate concentration of 0.02-0.08 m g / m l in buffer B was incubated at 20 ° C for 30 rain. The reaction was terminated by addition of 0.2 ml of 5% trichloroacetic acid and after centrifugation the supernatant was diluted with an equal volume of 2 M NaOH. After 10 min, the reaction products were determined by recording the absorbance at 440 nm. 1 unit of activity is defined as the amount of enzyme required to cause a change of absorbancy at 440 nm by 1 unit, and the specific activity is expressed as activity unit per milligram of the enzyme. (b) For amidase activity with Suc-(Ala)3-pNA, the assays were prepared in buffer B with a final concentration of 5 /~g/ml purified proteinase K, and substrate concentrations of 0.1 mmol/1 to 2.0 mmol/1. Assays of 1 ml vol. were incubated at 20 ° C for 1 h and the reaction was stopped by addition of 0.2 ml of glacial acetic acid and the absorbancy of the liberated nitroaniline was measured at 410 nm. (For comparative studies, the amidase activity was followed using Suc-(Ala)2-pNA as a substrate.) 1 unit of activity is expressed as the amount of enzyme which can liberate 1 mmol of p-nitroaniline per min of reaction (the amount of pnitroaniline was evaluated using a molar absorption coefficient, e = 9630 [1]). The specific activity is the number of activity units per mg of the enzyme. The change of absorbance at 410 nm (p-nitroaniline) and 440 nm (azoalbumin) during reaction time was plotted against substrate concentration as a general measure for the enzymatic activity of proteinase K. (c) To examine the influence of albumin on the enzymatic reaction with the synthetic substrate Suc-(Ala)3-pNA (because of the non-expected saturation behavior of azoalbumin, see Results), the experiments as described for azoalbumin were carried out with albumin but not terminated. After 30 rain of reaction, I ml of a mixture of the two substrates albumin and Suc-(Ala)3-pNA was added to every assay so that the albumin concentration remained 0.2-0.8 m g / m l , and a final Suc-(Ala)3-pNA concentration of 0.75 m M and a final enzyme concentration of 5 ~tg per ml of the assay mixture were achieved. After additional 1 h reaction time
178
0.4 ml of glacial acetic acid was added per assay and all assays were further treated as described for amidase activity. (d) The experiment with azoalbumin was repeated under exactly the same conditions as described in (a) but with subtilisin BPN' (obtained from Serva, Heidelberg, F.R.G.) instead of proteinase K. As references, equivalent assays were prepared and incubated but without albumin as substrate.
Results
A utodigestion studies Autodigestion of proteinase K was studied at pH 8.0 at three different concentrations, 0.01, 1.0 and 100 mg/ml. The enzyme sample was incubated in buffer B at room temperature. Aliquots were taken from the incubated enzyme sample at an appropriate time interval and assayed with Suc-(Ala)a-pNA as described above. In the assay mixture, the enzyme concentration was kept constant by diluting the 1.0 and 100 mg/ml aliquots by factors of 102 and 104 , respectively.
Purification The commercially available proteinase K showed two distinct peaks in the gelfiltration on a Sephadex G-75 column (Fig. 1). Fractions of the first symmetrical peak contained the total activity that was applied onto the column and the pooled material of this peak showed a single band in polyacrylamide gel electrophoresis at pH 4.5. When the same material was subjected to polyacrylamide gel electrophoresis in the presence of SDS, it showed several bands similar to the commercial product. When the commercial proteinase K preparation was exhaustively dialysed against 0.1 M Tris-HC1 (pH 8.0) prior to gelfiltration, the second peak was strongly reduced in the chromatogram.
Assay in the presence of methanol To follow the effect of methanol on the activity of proteinase K, the enzyme was incubated at 20°C in buffer B containing 20-80% methanol and assayed with Suc-(Ala)a-pNA dissolved in the same buffer. Temperature optimum The activity of proteinase K as a function of temperature was studied with Suc-(Ala)3-pNA as substrate in the range 4-70 °C in the absence and in the presence of 20% methanol. Inhibition studies Inhibition of the activity of proteinase K was assayed with Z-Ala-Ala-CK, Z-Ala-Phe-CK, and Z-Phe-CK in presence of methanol because these chloromethyl ketone analogs are almost insoluble in aqueous medium. A stock solution of each of the inhibitors was prepared in 50 mM Tris-HC1 (pH 8.0) containing 50% methanol. In the assay mixture, the inhibitor solution was added in up to 25 molar excess relative to proteinase K, and a final concentration of 5% of methanol was maintained. Turbidimetric measurements showed that in this low concentration range all three inhibitors were completely dissolved.
In SDS-polyacrylamide gel electrophoresis commercially available proteinase K showed one major band corresponding to about 30 kDa, and some minor bands around 10 kDa and less. In absence of SDS the same material also yielded more than one band in polyacrylamide gel electrophoresis at pH 4.5.
A utodigestion The autodigestion of proteinase K was monitored by measuring its residual activity after different activation periods. Within 5 h of activation, no reduction in the activity was observed if the enzyme concentration was in the range 0.01-100
12
<
O6
20
30
40 Fraction
50
60
70
no
Fig. 1. Elution profile of the Sephadex G-75 gel filtration of proteinase K. Elution buffer A with addition of 10 mM NaC1. Only the fractions of the first (symmetrical) peak showed proteolytic activity.
179 02
08
- -o .....
• ..........
a_
9 02
?
o
+o
2o
3b
do
I
dD
Time (h)
Fig. 2. Autodigestion of proteinase K as a function of dilution. Three proteinase K solutions were prepared with concentrations of 100, 1 and 0.01 mg/ml of the enzyme in buffer B, and incubated at 25 o C. At appropriate times of incubation, aliquots were taken to determine the enzymatic activity. All assays were prepared with a constant substrate concentration of 0.75 mM Suc-(Ala)3-pNA and an enzyme concentration of 14/~g per ml of the assay mixture. (a) Enzyme concentration of 1 and 100 mg/ml in the incubation mixture. (b) Enzyme concentration of 0.01 mg/ml proteinase K.
01
0.1 Substrate
Or5
0i.8
concentration
(g.1-1)
Fig. 3. Variation of substrate concentration with azoalbumin as substrate. Assays were prepared in buffer B with a constant proteinase K concentration of 10 /~g per ml of the assay mixture. The time of reaction was 30 min of 20 °C for each data point.
Temperature optimum m g / m l . E n z y m e s a m p l e s with high c o n c e n t r a t i o n s of 1 a n d 100 m g / m l showed n o loss of activity even after 48 h of activation (Fig. 2). If the enz y m e c o n c e n t r a t i o n was low, 0.01 m g / m l , a constant slow decrease in activity was o b s e r v e d after 5 h of activation o f the enzyme s a m p l e at r o o m temperature.
Substrate saturation curve and specific activity N o r m a l s a t u r a t i o n behavior, a h y p e r b o l a , was o b s e r v e d w h e n p r o t e i n a s e K was assayed with the synthetic s u b s t r a t e s S u c - ( A l a ) n - p N A , n = 2 a n d 3. I n contrast, with a z o a l b u m i n as substrate, the activity r e a c h e d a m a x i m u m at 0.2 g / 1 a n d then d e c r e a s e d (Fig. 3). T h e s a m e effect was o b s e r v e d w h e n the e x p e r i m e n t was c a r r i e d out with subtilisin B P N ' , T h e m a x i m u m activity of subtilisin B P N ' was achieved at 0 . 2 g l a z o a l b u m i n . A f t e r p r e i n c u b a t i o n for 30 m i n in presence of 0 . 2 - 0 . 8 g a l b u m i n / l , the e n z y m a t i c r e a c t i o n of p r o t e i n a s e K a n d subtilisin B P N ' with S u c - ( A l a ) 3 - p N A was n o t affected. C o n s i d e r i n g the linear regions of the activity curves, the specific activity of p r o t e i n a s e K was c a l c u l a t e d as 0.8 u n i t s / m g with a z o a l b u m i n , as 13 units/mg w i t h S u c - ( A l a ) 3 - p N A a n d as 3.5 u n i t s / m g with S u c - ( A l a ) 2 - p N A . T h e specific activity of p r o t e i n a s e K against the synthetic substrate was n o t r e d u c e d after p r e i n c u b a t i o n in presence of a l b u m i n as d e s c r i b e d above.
A l t h o u g h there was a t o t a l loss of a p p r o x . 40% of the activity of p r o t e i n a s e K w h e n m e t h a n o l was a d d e d , the overall b e h a v i o r of the e n z y m e with the v a r i a t i o n o f t e m p e r a t u r e was n e a r l y the s a m e in p r e s e n c e o r in a b s e n c e of m e t h a n o l . A t 4 o C, b o t h activities are c o m p a r a b l e a n d with increasing temp e r a t u r e the r e a c t i o n r a t e increases m o r e r a p i d l y in a b s e n c e t h a n in p r e s e n c e of m e t h a n o l (Fig. 4). U n d e r b o t h c o n d i t i o n s , m a x i m u m activity was
09
/O~ . O _ _ o _ _ _ O ~
08
~o/
07
/
06
.]
"o a
/e/ o~O/'-
o
0 5
//
<'~
o4
~O,/~ . o
o+
~o,,. O\ ~o,,
~O j
O ~ O ~
,~-~
0,.d~
"",,0
02 01
~
2b
3'o
4b
~o
+'o
¢o
Temperature °C
Fig. 4. Temperature optimum of proteinase K. The temperature optimum was determined without methanol (a), and in presence of 20% methanol (b) in the range of 4-70 °C using Suc-(Ala)3-pNA as substrate. The tests were carried out with a constant substrate concentration of 0.75 mM and a constant proteinase K concentration of 7 #g/ml in buffer B. Enzyme and substrate were equilibrated at the reaction temperature for 15 min before assaying. After incubation, the reaction was started and terminated after 60 min as described under Materials and Methods.
180
°75~o,,_
o b t a i n e d at 3 7 ° C a n d r e m a i n e d m o r e o r less c o n s t a n t until 5 5 ° C . A t 7 0 ° C , the activity of p r o t e i n a s e K was r e d u c e d to 67 a n d 59%, respectively, c o m p a r e d to the m a x i m u m activity (at 37 o C) in the a b s e n c e a n d presence of m e t h a n o l .
070~
~X
0 65t/e e\e~._
Enzyme inhibition Methanol inhibition.
I n p r e s e n c e o f 10% methanol. Proteinase K still exhibits 90% of its original activity which finally d e c r e a s e d to only 10% in presence o f 40% m e t h a n o l . LineweaverBurk s u b s t r a t e s a t u r a t i o n curves were t a k e n in aqueous m e d i u m a n d in presence of 10, 20 a n d 30% methanol. F r o m the c o r r e s p o n d i n g d o u b l e - r e ciprocal plots (Fig. 5), K m values were c a l c u l a t e d as 0.77 m m o l / 1 w i t h o u t m e t h a n o l , 1.11 m m o l / 1 with 10%, 2.33 m m o l / 1 with 20% a n d 7.69 m m o l / 1 with 30% m e t h a n o l . Synthetic inhibitors. The i n h i b i t o r y activities o f b o t h the c h l o r o m e t h y l k e t o n e derivatives Z - A l a A l a - C K a n d Z - A l a - P h e - C K are c o m p a r a b l e with a m a x i m u m i n h i b i t i o n of a b o u t 50% at an i n h i b i t o r c o n c e n t r a t i o n c o r r e s p o n d i n g to 10 m o l a r excess relative to p r o t e i n a s e K. U n d e r the s a m e c o n d i tions of m e a s u r e m e n t , the i n h i b i t i o n of p r o t e i n a s e o d
/O c • e
o ~1<~
-1
~"
e~
./
.
O'
0 [Substrate] 1,
~"
e-°"
J 2
Inhtbitor
J 3
J 4
i 5
concentr'Qtion
I 6
I 7
I 8
(lO-SmM)
Fig. 6. Activity of proteinase K in presence of chloromethyl ketone derivatives. Inhibition of activity of proteinase K was assayed under the same experimental conditions as described for Fig. 4 at 25 ° C. Before adding the substrate, proteinase K was incubated for 5 min in the presence of a chloromethyl ketone derivative. The final methanol concentration in every assay was 5% and the inhibitor concentration was increased up to 25-fold molar excess relative to proteinase K. (a) Activity of proteinase K in presence of Z-Phe-CK. As the inhibition rate of Z-Ala-Ala-CK and Z-Ala-Phe-CK were in the same range the activity of proteinase K in the presence of only Z-Ala-PheCK is shown in (b). K b y Z - P h e - C K was o n l y a b o u t 20%. Even at 25-fold m o l a r excess of each of the three chlorom e t h y l k e t o n e derivatives, the i n h i b i t i o n of p r o teinase K was n o t i n c r e a s e d further (Fig. 6).
Discussion
~'~
O"
1
I 1
"Oh
¢
• •
-
¢
b
30/ 0
2
] mmol-1
Fig. 5. Effect of methanol on the activity of proteinase K. Reactions were carried out at 20 °C with 0.75 mM Suc-(Ala)3pNA as substrate and 5 #g purified proteinase K per ml of the assay mixture using buffer B, containing 0-30% methanol. Substrate and enzyme were incubated for 10 rain in the buffer containing the same methanol concentration as the final assay mixture before use. (a) 0% methanol, (b) 10% methanol, (c) 20% methanol, and (d) 30% methanol. Substrate saturation curves were taken and the corresponding double-reciprocal plots are shown. The functions (a-d) represent the same experimental data but with different concentrations of methanol. K m values a r e ( a ) 0.77 mmol/l, (b) 1.11 mmol/1, (c) 2.33 mmol/1, and (d) 7.69 mmol/1.
T h e c o m m e r c i a l s a m p l e s of the very p o t e n t p r o t e o l y t i c e n z y m e p r o t e i n a s e K c o n t a i n autolysis p r o d u c t s w h i c h are largely r e m o v e d b y dialysis a n d c o m p l e t e l y b y gel filtration (Fig. 1). A s p r o teinase K is active even in the p r e s e n c e o f S D S [1], the p u r e e n z y m e r a p i d l y autolyses u n d e r r o u t i n e c o n d i t i o n s for S D S - g e l electrophoresis which inc l u d e b o i l i n g in the p r e s e n c e of 1% SDS. T h u s gel electrophoresis with a n d w i t h o u t S D S of c o m m e r cial s a m p l e s s h o w e d m i n o r b a n d s suggesting cleavage p r o d u c t s less t h a n 10 k D a . A f t e r gel filtration, the s a m e gel p a t t e r n was o b t a i n e d with S D S gel electrophoresis, b u t in a b s e n c e of S D S o n l y a single b a n d was o b t a i n e d a c c o r d i n g to a m o l e c u l a r weight of 30 k D a . A u t o l y s i s is v i r t u a l l y a b s e n t over a p e r i o d of at least 48 h if the c o n c e n t r a t i o n o f p r o t e i n a s e K is at or a b o v e 1 m g / m l , b u t occurs slowly after a lag
181 period of 2 h in dilute aqueous solutions at or below 0.01 mg/ml (Fig. 2). This observation is consistent with crystallographic studies which showed that proteinase K crystallizes without cleavage from highly concentrated solutions of 10 mg/ml. In the crystal lattice, the proteinase K molecules form dimers related by 2-fold symmetry axes [4], and it appears that this or a comparable aggregation also occurs in solution above a certain concentration, thereby preventing autolysis. We observe a relation between autolysis rate and decrease of enzymatic activity with a lag period of 2 h in dilute solution of proteinase K. This suggests that the first cuts do not influence the activity and that it takes some time before the structure of the active site, i.e., the.substrate recognition site a n d / o r the catalytically active Asp-39-His69-Ser-224 triad, is actually changed. Obviously proteinase K autolyses if the native and very globular tertiary structure [4] is changed by denaturing conditions that induce more or less unfolding of the enzyme. The oxidized insulin B chain was previously used as a natural substrate for proteinase K in order to study cleavage specificity [6]. We employed azoalbumin as natural substrate and found an interesting substrate saturation curve (Fig. 3) where the enzymatic activity against azoalbumin decreases strongly when the molar ratio azoalbumin/proteinase K is raised to 20 : 1. This behavior could be due to product inhibition but the experiments where Suc-(Ala)3-pNA as a second substrate is added and readily cleaved even after preincubation with albumin clearly indicate that proteinase K is not product-inhibited. We rather have to assume that non-specific protein-protein interaction of albumin molecules (when increasing the albumin concentration) results in more resistance towards cleavage by proteinase K. This interpretation is supported by the same experiments with subtilisin BPN' instead of proteinase K which showed comparable results. The observed effect in substrate saturation curve with azoalbumin as substrate is not specific for proteinase K. Investigations with synthetic inhibitors and substrates illustrated that the binding to the active site of proteinase K is dependent on peptide chain length. First this is evident from the specific activities of proteinase K against Suc-(Ala)3-pNA , 13
units/mg, and against the shorter analogue Suc(Ala)2-pNA , 3.5 units/mg. It has been shown recently [7] that the activity of proteinase K against different synthetic substrates is lower compared to the related enzymes subtilisin BPN' and thermitase, although proteinase K displays a higher activity against naturally occurring substrates. The same influence of peptide chain length was found with symhetic peptide chioromethyl ketone inhibitors, a class of substances which are used to inhibit serine proteinases [8], because they react covalently with the active-site serine and histidine side-chains. The inhibitory action of these compounds for proteinase K is not very efficient, since in no case did the maximum inhibition exceed the 50% level. The inhibition is comparable for Z-AlaAla-CK and Z-Ala-Phe-ZK, but is especially poor for the shorter analogue Z-Phe-CK where the inhibition was only about 10%. This weak effect is probably due to poor binding of the inhibitor to the substrate-binding site which involves the formation of a pleated sheet structure between the substrate (inhibitor) and two strands of proteinase K and is most favorable for dipeptides or even longer peptides. The influence of methanol on the activity of proteinase K has been studied because we tried to use methanol as a cryosolvent in low temperature crystallographic studies of the enzyme. In assays for enzyme action K m increases with the addition of methanol and, in contrast, the Vma~ are identical in aqueous solution and in presence of methanol (Fig. 5). This result clearly indicates the existence of classical competitive inhibition. Considering the general enzymatic mechanism of serine proteinases with the formation of the acyl enzyme in the first, and the hydrolysis of the acyl enzyme in the second step of catalysis one could assume that methanol competes with water in the second step with the interesting consequence of a trapped transition state complex in presence of methanol even at room temperature. However, according to the ping-pong mechanism proposed for the enzymatic mechanism of serine proteinases (Cleland, 1970) a competitor of the second substrate water would exhibit a non-competitive type of inhibition toward the first substrate Suc-(Ala)a-pNA. Obviously, the formation of the enzyme-substrate complex is affected by methanol which successfully
182
/~sn161~~
~
Fig. 7. Diagram of the active site region in proteinase K, based on the 1.5 ,A resolution X-ray analysis. Side-chains of the active triad, Asp-39-His-69-Ser-224, are drawn solid, and hydrogen bonds by dashed lines. Water molecules are designated OW, the more important water molecules OW328 ( = OW *) and OW335 ( = O W * * ) are filled. We are grateful to Dr. Betzel for permission to reproduce this figure from Ref. 4.
competes for the substrate-binding site because it is in large molecular excess (methanol/Suc(Ala)3-pNA is 4000:1 for 10% methanol).Fig. 4 illustrates the activity of proteinase K as a function of temperature in absence and in presence of 20% methanol. The effect of methanol is negligible at 4%, but it then continuously decreases the activity to about 50% at the temperature optimum, 37 o C. The maximum activity of proteinase K is in the range 37-55 o C. At 70°C, more than 50% of the maximum activity is still retained, indicating a thermal stability in the range as observed for wildtype subtilisin 9. The influence of methanol on the activity of proteinase K can be discussed on the basis of the structure of the active site, which is known to atomic detail from the X-ray analysis. As shown in Fig. 7, the catalytic triad, Asp-38-His-69-Ser224, is in hydrogen bonding contact with water molecules, and several other water molecules hydrogen are bound to the substrate recognition
region (that begins at Ser-132, the beginning of the second strand of the recognition region, Gly-100, is located on the right above Asp-39, not shown in Fig. 7). We can put forward the hypothesis that it is the replacement of water molecules by methanol which competes with substrate binding, thereby increasing the K m. It could be that (i) some or all water molecules in the active site are replaced and (ii) that those water molecules probably involved in the catalytic mechanism are replaced. These are OW335 which is located in the oxyanion hole and OW328 which keeps Asp-39 in the correct conformation so that the Asp 39---His-69 hydrogen bond can form. If these water molecules are replaced by methanol, direct interference with the substrate binding can be anticipated. This holds especially for OW335 whose substitution with methanol would reduce or inhibit binding of the tetrahedral transition intermediate which requires this site for charge delocalization. We could also assume (iii) that methanol binds by hydrogen bonding to Ser-224 and by hydrophobic interactions to the imidazole of His-69, thereby impeding proper binding of the substrate molecules.
Acknowledgement We are grateful to Dr. Burgert, Institut fiar Organische Chemie der Freien Universit~it Berlin, and to Prof. K.-D. Jany, Institut fiir Organische Chemie und Biochemie der Universitat Darmstadt, for providing samples of peptide chloromethyl ketone inhibitors.
References 1 Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H.D. and Land, H. (1974) Eur. J. Biochem. 47, 91-97. 2 PRhler, A., Banerjee, A., Dattagupta, J.K., Fujiwara, T., Lindner, K., Pal, G.P., Suck, D., Weber, G. and Saenger, W. (1984) EMBO J. 3, 1311-1313. 3 Jany, K.D., Lederer, G. and Mayer, B. (1986) FEBS Lett. 199, 139-144. 4 Betzel, C. (1986) Ph.D. thesis, Freie Universit~it Berlin. 5 Laemmli, U.K. (1970) Nature 227, 680-685. 6 Kraus, E., Kiltz, H.-H. and Femfert, U. (1976) HoppeSeyler's Z. Physiol. Chem. 357, 233-237. 7 BrSmme, D., Peters, K., Fink, S. and Fittkau, S. (1986) Arch. Biochem. Biophys. 244, 2, 439-446. 8 Shaw, E. (1970) The Enzymes (Boyer, P., ed.), 3rd Edn., p. 91, Academic Press, New York. 9 Bryan, P.N., Rollence, M.L., Pantoliano, M.W., Wood, J., Finzel, B.C., Gilliland, G.L., Howard, A.J. and Poulos, T.L. (1986) Proteins 1, 4, 326-334.
Biochimica et Biophysica Acta 954 (1988) 176-182
Elsevier BBA33129
Autolysis and inhibition of proteinase K, a subtilisin-related serine proteinase isolated from the fungus Tritirachium album Limber
Ji~rgen Bajorath, Wolfram Saenger and Gout Pada Pal * Institut ]'dr Kristallographie, Freie Universitiit Berlin, Berlin (F.R.G.)
(Received 13 October 1987) Key words: ProteinaseK; Serineproteinase; Protein-protein interaction; Autodigestion; Enzymeinhibition; Syntheticinhibitor; (T. album) The activity of proteinase K (EC 3.4.21.14), a subtilisin-related serine proteinase, was assayed with azoalbumin that showed non-expected behavior in substrate saturation curve because of interaction between albumin molecules. Succinyl-(Aia)n-p-nitroanilide with n = 2 and 3, yielded specific activities of 3.5, 13 u n i t s / r a g protein, respectively, reflecting a chain length dependence. The influence of peptide chain length on binding to proteinase K was also observed using mono- and dipeptide chloromethyi ketone inhibitors. They showed a maximum inhibition. They showed a maximum inhibition of proteinase K in solution of only about 50% even at a more than 20-fold molar excess. With the above substrates, the Vmax is not affected in presence of 10, 20 and 30% methanol, but the K m differs remarkably, suggesting competitive inhibition. The activity of proteinase K shows a maximum at 37°C, and a temperature profile with more than 80% maximum activity in the range 20-60 o C. Autolysis of the enzyme is observed during sample preparation for SDS-gel electrophoresis and at low concentration (0.01 m g / m l ) in aqueous solution. It does not o c c u r at higher proteinase K concentrations at or above 1.0 m g / m l , consistent with crystallographic studies. Introduction
Proteinase K (EC, 3.4.21.14) isolated from fungus, Tritirachium album Limber, is one of the most active serine proteinases. It can hydrolyse even native keratin and remains active in presence
Abbreviations: Suc-(Ala)3-pNA, succinylalanylalanylalanylpara-nitroanilide; Suc-(Ala)2-pNA, succinylalanylalanyl-para-
nitroanilide; Z-Ala-Phe-CK, carbobenzoxyalanylphenyl chloromethyl ketone; Z-Ala-Ala-CK, carbobenzoxyalanylalanyl chloromethylketone; Z-Phe-CK, carbobenzoxyphenyl chloromethylketone. * Present address: European Molecular Biology Laboratory, Heidelberg, F.R.G. Correspondence:J. Bajorath, Institut ftir KristaUographie,Freie Universit~it Berlin, TakustraBe6, D-1000, Berlin, F.R.G.
of sodium dodecyl sulfate and urea [1]. The enzyme shows an unusual retention in a colunm of Sephadex G-75 which led to an erroneous moleculax weight determination of 18500 [1]. After the elucidation of its three-dimensional structure, the molecular weight of proteinase K was calculated to 27000 [2] in good agreement with the value determined by SDS-polyacrylamide gel electrophoresis [2]. Recently, the true molecular weight has been evaluated as 28 930 from the amino-acid sequence [3]. Although the three-dimensional structure of proteinase K has been refined at 1.5 resolution by X-ray crystallographic methods [4] and is thus known in great detail, the biochemical properties of the enzyme are not yet well characterized. The present studies on the biochemical behavior of proteinase K should help to evaluate further the structure-function relationship of this enzyme.
0167-4838/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
177
Materials and Methods
Materials Proteinase K was purchased from E. Merck, Darmstadt, F.R.G.; azoalbumin from Sigma Chemicals; albumin from Boehringer, Mannheim; Suc-(Ala),-pNA from Serva Feinbiochemica, Heidelberg, F.R.G. The synthetic inhibitor, Z-AlaPhe-CK, was supplied by Dr. Burgert, Institut fiir Organische Chemie, Freie Universit~it Berlin, ZAla-Ala-CK and Z-Phe-CK were obtained from Prof. Jany, Universit~t Stuttgart. All other commonly available reagents were of analytical grade. For characterization of commercial proteinase K, standard polyacrylamide gel electrophoresis was carried out at pH 7.2 as described [5]. The material was pretreated with SDS and 2-mercaptoethanol. In absence of SDS, polyacrylamide gel electrophoresis was performed at pH 4.3. Buffers A and B were prepared according to Ebeling et al. [1]. Buffer A: 0.01 M Tris-HC1, 5 mM CaC12 (pH 8.0); and buffer B: 50 mM TrisHC1, 5 mM CaC12 (pH 8.0).
Purification of proteinase K A 10% aqueous solution of the commercial product was subjected to gel-filtration using an Sephadex G-75 column (2 x 70 cm) which was pre-equilibrated with buffer A at room temperature. The protein was eluted with the same buffer at a flow rate of approx. 40 m l / h and fractions of 6 ml were collected. The presence of protein was monitored by absorbance measurements at 280 nm and the proteinase K activity was assayed with azoalbumin as substrate. Active fractions were pooled, dialysed exhaustively against 1 m M calcium acetate at 4 ° C , and lyophilized. The material was stored at 4 ° C till further use.
Assay of enzyme activity The proteolytic activity of proteinase K was monitored using azoalbumin as substrate, and for the routine assay its amidase activity was followed using Suc-(Ala)3-pNA as substrate. Prior to the assay, lyophilized proteinase K was dissolved at a concentration of 1 m g / m i in buffer B and incubated routinely at 20 ° C for 10 min.
Substrate saturation curve For a data point on the substrate saturation
curve, experiments were performed with azoalbumin and Suc-(Ala)3-pNA as substrates. (a) Azoalbumin. 1 ml of the assay mixture containing 10 ~tg of the enzyme and a final substrate concentration of 0.02-0.08 m g / m l in buffer B was incubated at 20 ° C for 30 rain. The reaction was terminated by addition of 0.2 ml of 5% trichloroacetic acid and after centrifugation the supernatant was diluted with an equal volume of 2 M NaOH. After 10 min, the reaction products were determined by recording the absorbance at 440 nm. 1 unit of activity is defined as the amount of enzyme required to cause a change of absorbancy at 440 nm by 1 unit, and the specific activity is expressed as activity unit per milligram of the enzyme. (b) For amidase activity with Suc-(Ala)3-pNA, the assays were prepared in buffer B with a final concentration of 5 /~g/ml purified proteinase K, and substrate concentrations of 0.1 mmol/1 to 2.0 mmol/1. Assays of 1 ml vol. were incubated at 20 ° C for 1 h and the reaction was stopped by addition of 0.2 ml of glacial acetic acid and the absorbancy of the liberated nitroaniline was measured at 410 nm. (For comparative studies, the amidase activity was followed using Suc-(Ala)2-pNA as a substrate.) 1 unit of activity is expressed as the amount of enzyme which can liberate 1 mmol of p-nitroaniline per min of reaction (the amount of pnitroaniline was evaluated using a molar absorption coefficient, e = 9630 [1]). The specific activity is the number of activity units per mg of the enzyme. The change of absorbance at 410 nm (p-nitroaniline) and 440 nm (azoalbumin) during reaction time was plotted against substrate concentration as a general measure for the enzymatic activity of proteinase K. (c) To examine the influence of albumin on the enzymatic reaction with the synthetic substrate Suc-(Ala)3-pNA (because of the non-expected saturation behavior of azoalbumin, see Results), the experiments as described for azoalbumin were carried out with albumin but not terminated. After 30 rain of reaction, I ml of a mixture of the two substrates albumin and Suc-(Ala)3-pNA was added to every assay so that the albumin concentration remained 0.2-0.8 m g / m l , and a final Suc-(Ala)3-pNA concentration of 0.75 m M and a final enzyme concentration of 5 ~tg per ml of the assay mixture were achieved. After additional 1 h reaction time
178
0.4 ml of glacial acetic acid was added per assay and all assays were further treated as described for amidase activity. (d) The experiment with azoalbumin was repeated under exactly the same conditions as described in (a) but with subtilisin BPN' (obtained from Serva, Heidelberg, F.R.G.) instead of proteinase K. As references, equivalent assays were prepared and incubated but without albumin as substrate.
Results
A utodigestion studies Autodigestion of proteinase K was studied at pH 8.0 at three different concentrations, 0.01, 1.0 and 100 mg/ml. The enzyme sample was incubated in buffer B at room temperature. Aliquots were taken from the incubated enzyme sample at an appropriate time interval and assayed with Suc-(Ala)a-pNA as described above. In the assay mixture, the enzyme concentration was kept constant by diluting the 1.0 and 100 mg/ml aliquots by factors of 102 and 104 , respectively.
Purification The commercially available proteinase K showed two distinct peaks in the gelfiltration on a Sephadex G-75 column (Fig. 1). Fractions of the first symmetrical peak contained the total activity that was applied onto the column and the pooled material of this peak showed a single band in polyacrylamide gel electrophoresis at pH 4.5. When the same material was subjected to polyacrylamide gel electrophoresis in the presence of SDS, it showed several bands similar to the commercial product. When the commercial proteinase K preparation was exhaustively dialysed against 0.1 M Tris-HC1 (pH 8.0) prior to gelfiltration, the second peak was strongly reduced in the chromatogram.
Assay in the presence of methanol To follow the effect of methanol on the activity of proteinase K, the enzyme was incubated at 20°C in buffer B containing 20-80% methanol and assayed with Suc-(Ala)a-pNA dissolved in the same buffer. Temperature optimum The activity of proteinase K as a function of temperature was studied with Suc-(Ala)3-pNA as substrate in the range 4-70 °C in the absence and in the presence of 20% methanol. Inhibition studies Inhibition of the activity of proteinase K was assayed with Z-Ala-Ala-CK, Z-Ala-Phe-CK, and Z-Phe-CK in presence of methanol because these chloromethyl ketone analogs are almost insoluble in aqueous medium. A stock solution of each of the inhibitors was prepared in 50 mM Tris-HC1 (pH 8.0) containing 50% methanol. In the assay mixture, the inhibitor solution was added in up to 25 molar excess relative to proteinase K, and a final concentration of 5% of methanol was maintained. Turbidimetric measurements showed that in this low concentration range all three inhibitors were completely dissolved.
In SDS-polyacrylamide gel electrophoresis commercially available proteinase K showed one major band corresponding to about 30 kDa, and some minor bands around 10 kDa and less. In absence of SDS the same material also yielded more than one band in polyacrylamide gel electrophoresis at pH 4.5.
A utodigestion The autodigestion of proteinase K was monitored by measuring its residual activity after different activation periods. Within 5 h of activation, no reduction in the activity was observed if the enzyme concentration was in the range 0.01-100
12
<
O6
20
30
40 Fraction
50
60
70
no
Fig. 1. Elution profile of the Sephadex G-75 gel filtration of proteinase K. Elution buffer A with addition of 10 mM NaC1. Only the fractions of the first (symmetrical) peak showed proteolytic activity.
179 02
08
- -o .....
• ..........
a_
9 02
?
o
+o
2o
3b
do
I
dD
Time (h)
Fig. 2. Autodigestion of proteinase K as a function of dilution. Three proteinase K solutions were prepared with concentrations of 100, 1 and 0.01 mg/ml of the enzyme in buffer B, and incubated at 25 o C. At appropriate times of incubation, aliquots were taken to determine the enzymatic activity. All assays were prepared with a constant substrate concentration of 0.75 mM Suc-(Ala)3-pNA and an enzyme concentration of 14/~g per ml of the assay mixture. (a) Enzyme concentration of 1 and 100 mg/ml in the incubation mixture. (b) Enzyme concentration of 0.01 mg/ml proteinase K.
01
0.1 Substrate
Or5
0i.8
concentration
(g.1-1)
Fig. 3. Variation of substrate concentration with azoalbumin as substrate. Assays were prepared in buffer B with a constant proteinase K concentration of 10 /~g per ml of the assay mixture. The time of reaction was 30 min of 20 °C for each data point.
Temperature optimum m g / m l . E n z y m e s a m p l e s with high c o n c e n t r a t i o n s of 1 a n d 100 m g / m l showed n o loss of activity even after 48 h of activation (Fig. 2). If the enz y m e c o n c e n t r a t i o n was low, 0.01 m g / m l , a constant slow decrease in activity was o b s e r v e d after 5 h of activation o f the enzyme s a m p l e at r o o m temperature.
Substrate saturation curve and specific activity N o r m a l s a t u r a t i o n behavior, a h y p e r b o l a , was o b s e r v e d w h e n p r o t e i n a s e K was assayed with the synthetic s u b s t r a t e s S u c - ( A l a ) n - p N A , n = 2 a n d 3. I n contrast, with a z o a l b u m i n as substrate, the activity r e a c h e d a m a x i m u m at 0.2 g / 1 a n d then d e c r e a s e d (Fig. 3). T h e s a m e effect was o b s e r v e d w h e n the e x p e r i m e n t was c a r r i e d out with subtilisin B P N ' , T h e m a x i m u m activity of subtilisin B P N ' was achieved at 0 . 2 g l a z o a l b u m i n . A f t e r p r e i n c u b a t i o n for 30 m i n in presence of 0 . 2 - 0 . 8 g a l b u m i n / l , the e n z y m a t i c r e a c t i o n of p r o t e i n a s e K a n d subtilisin B P N ' with S u c - ( A l a ) 3 - p N A was n o t affected. C o n s i d e r i n g the linear regions of the activity curves, the specific activity of p r o t e i n a s e K was c a l c u l a t e d as 0.8 u n i t s / m g with a z o a l b u m i n , as 13 units/mg w i t h S u c - ( A l a ) 3 - p N A a n d as 3.5 u n i t s / m g with S u c - ( A l a ) 2 - p N A . T h e specific activity of p r o t e i n a s e K against the synthetic substrate was n o t r e d u c e d after p r e i n c u b a t i o n in presence of a l b u m i n as d e s c r i b e d above.
A l t h o u g h there was a t o t a l loss of a p p r o x . 40% of the activity of p r o t e i n a s e K w h e n m e t h a n o l was a d d e d , the overall b e h a v i o r of the e n z y m e with the v a r i a t i o n o f t e m p e r a t u r e was n e a r l y the s a m e in p r e s e n c e o r in a b s e n c e of m e t h a n o l . A t 4 o C, b o t h activities are c o m p a r a b l e a n d with increasing temp e r a t u r e the r e a c t i o n r a t e increases m o r e r a p i d l y in a b s e n c e t h a n in p r e s e n c e of m e t h a n o l (Fig. 4). U n d e r b o t h c o n d i t i o n s , m a x i m u m activity was
09
/O~ . O _ _ o _ _ _ O ~
08
~o/
07
/
06
.]
"o a
/e/ o~O/'-
o
0 5
//
<'~
o4
~O,/~ . o
o+
~o,,. O\ ~o,,
~O j
O ~ O ~
,~-~
0,.d~
"",,0
02 01
~
2b
3'o
4b
~o
+'o
¢o
Temperature °C
Fig. 4. Temperature optimum of proteinase K. The temperature optimum was determined without methanol (a), and in presence of 20% methanol (b) in the range of 4-70 °C using Suc-(Ala)3-pNA as substrate. The tests were carried out with a constant substrate concentration of 0.75 mM and a constant proteinase K concentration of 7 #g/ml in buffer B. Enzyme and substrate were equilibrated at the reaction temperature for 15 min before assaying. After incubation, the reaction was started and terminated after 60 min as described under Materials and Methods.
180
°75~o,,_
o b t a i n e d at 3 7 ° C a n d r e m a i n e d m o r e o r less c o n s t a n t until 5 5 ° C . A t 7 0 ° C , the activity of p r o t e i n a s e K was r e d u c e d to 67 a n d 59%, respectively, c o m p a r e d to the m a x i m u m activity (at 37 o C) in the a b s e n c e a n d presence of m e t h a n o l .
070~
~X
0 65t/e e\e~._
Enzyme inhibition Methanol inhibition.
I n p r e s e n c e o f 10% methanol. Proteinase K still exhibits 90% of its original activity which finally d e c r e a s e d to only 10% in presence o f 40% m e t h a n o l . LineweaverBurk s u b s t r a t e s a t u r a t i o n curves were t a k e n in aqueous m e d i u m a n d in presence of 10, 20 a n d 30% methanol. F r o m the c o r r e s p o n d i n g d o u b l e - r e ciprocal plots (Fig. 5), K m values were c a l c u l a t e d as 0.77 m m o l / 1 w i t h o u t m e t h a n o l , 1.11 m m o l / 1 with 10%, 2.33 m m o l / 1 with 20% a n d 7.69 m m o l / 1 with 30% m e t h a n o l . Synthetic inhibitors. The i n h i b i t o r y activities o f b o t h the c h l o r o m e t h y l k e t o n e derivatives Z - A l a A l a - C K a n d Z - A l a - P h e - C K are c o m p a r a b l e with a m a x i m u m i n h i b i t i o n of a b o u t 50% at an i n h i b i t o r c o n c e n t r a t i o n c o r r e s p o n d i n g to 10 m o l a r excess relative to p r o t e i n a s e K. U n d e r the s a m e c o n d i tions of m e a s u r e m e n t , the i n h i b i t i o n of p r o t e i n a s e o d
/O c • e
o ~1<~
-1
~"
e~
./
.
O'
0 [Substrate] 1,
~"
e-°"
J 2
Inhtbitor
J 3
J 4
i 5
concentr'Qtion
I 6
I 7
I 8
(lO-SmM)
Fig. 6. Activity of proteinase K in presence of chloromethyl ketone derivatives. Inhibition of activity of proteinase K was assayed under the same experimental conditions as described for Fig. 4 at 25 ° C. Before adding the substrate, proteinase K was incubated for 5 min in the presence of a chloromethyl ketone derivative. The final methanol concentration in every assay was 5% and the inhibitor concentration was increased up to 25-fold molar excess relative to proteinase K. (a) Activity of proteinase K in presence of Z-Phe-CK. As the inhibition rate of Z-Ala-Ala-CK and Z-Ala-Phe-CK were in the same range the activity of proteinase K in the presence of only Z-Ala-PheCK is shown in (b). K b y Z - P h e - C K was o n l y a b o u t 20%. Even at 25-fold m o l a r excess of each of the three chlorom e t h y l k e t o n e derivatives, the i n h i b i t i o n of p r o teinase K was n o t i n c r e a s e d further (Fig. 6).
Discussion
~'~
O"
1
I 1
"Oh
¢
• •
-
¢
b
30/ 0
2
] mmol-1
Fig. 5. Effect of methanol on the activity of proteinase K. Reactions were carried out at 20 °C with 0.75 mM Suc-(Ala)3pNA as substrate and 5 #g purified proteinase K per ml of the assay mixture using buffer B, containing 0-30% methanol. Substrate and enzyme were incubated for 10 rain in the buffer containing the same methanol concentration as the final assay mixture before use. (a) 0% methanol, (b) 10% methanol, (c) 20% methanol, and (d) 30% methanol. Substrate saturation curves were taken and the corresponding double-reciprocal plots are shown. The functions (a-d) represent the same experimental data but with different concentrations of methanol. K m values a r e ( a ) 0.77 mmol/l, (b) 1.11 mmol/1, (c) 2.33 mmol/1, and (d) 7.69 mmol/1.
T h e c o m m e r c i a l s a m p l e s of the very p o t e n t p r o t e o l y t i c e n z y m e p r o t e i n a s e K c o n t a i n autolysis p r o d u c t s w h i c h are largely r e m o v e d b y dialysis a n d c o m p l e t e l y b y gel filtration (Fig. 1). A s p r o teinase K is active even in the p r e s e n c e o f S D S [1], the p u r e e n z y m e r a p i d l y autolyses u n d e r r o u t i n e c o n d i t i o n s for S D S - g e l electrophoresis which inc l u d e b o i l i n g in the p r e s e n c e of 1% SDS. T h u s gel electrophoresis with a n d w i t h o u t S D S of c o m m e r cial s a m p l e s s h o w e d m i n o r b a n d s suggesting cleavage p r o d u c t s less t h a n 10 k D a . A f t e r gel filtration, the s a m e gel p a t t e r n was o b t a i n e d with S D S gel electrophoresis, b u t in a b s e n c e of S D S o n l y a single b a n d was o b t a i n e d a c c o r d i n g to a m o l e c u l a r weight of 30 k D a . A u t o l y s i s is v i r t u a l l y a b s e n t over a p e r i o d of at least 48 h if the c o n c e n t r a t i o n o f p r o t e i n a s e K is at or a b o v e 1 m g / m l , b u t occurs slowly after a lag
181 period of 2 h in dilute aqueous solutions at or below 0.01 mg/ml (Fig. 2). This observation is consistent with crystallographic studies which showed that proteinase K crystallizes without cleavage from highly concentrated solutions of 10 mg/ml. In the crystal lattice, the proteinase K molecules form dimers related by 2-fold symmetry axes [4], and it appears that this or a comparable aggregation also occurs in solution above a certain concentration, thereby preventing autolysis. We observe a relation between autolysis rate and decrease of enzymatic activity with a lag period of 2 h in dilute solution of proteinase K. This suggests that the first cuts do not influence the activity and that it takes some time before the structure of the active site, i.e., the.substrate recognition site a n d / o r the catalytically active Asp-39-His69-Ser-224 triad, is actually changed. Obviously proteinase K autolyses if the native and very globular tertiary structure [4] is changed by denaturing conditions that induce more or less unfolding of the enzyme. The oxidized insulin B chain was previously used as a natural substrate for proteinase K in order to study cleavage specificity [6]. We employed azoalbumin as natural substrate and found an interesting substrate saturation curve (Fig. 3) where the enzymatic activity against azoalbumin decreases strongly when the molar ratio azoalbumin/proteinase K is raised to 20 : 1. This behavior could be due to product inhibition but the experiments where Suc-(Ala)3-pNA as a second substrate is added and readily cleaved even after preincubation with albumin clearly indicate that proteinase K is not product-inhibited. We rather have to assume that non-specific protein-protein interaction of albumin molecules (when increasing the albumin concentration) results in more resistance towards cleavage by proteinase K. This interpretation is supported by the same experiments with subtilisin BPN' instead of proteinase K which showed comparable results. The observed effect in substrate saturation curve with azoalbumin as substrate is not specific for proteinase K. Investigations with synthetic inhibitors and substrates illustrated that the binding to the active site of proteinase K is dependent on peptide chain length. First this is evident from the specific activities of proteinase K against Suc-(Ala)3-pNA , 13
units/mg, and against the shorter analogue Suc(Ala)2-pNA , 3.5 units/mg. It has been shown recently [7] that the activity of proteinase K against different synthetic substrates is lower compared to the related enzymes subtilisin BPN' and thermitase, although proteinase K displays a higher activity against naturally occurring substrates. The same influence of peptide chain length was found with symhetic peptide chioromethyl ketone inhibitors, a class of substances which are used to inhibit serine proteinases [8], because they react covalently with the active-site serine and histidine side-chains. The inhibitory action of these compounds for proteinase K is not very efficient, since in no case did the maximum inhibition exceed the 50% level. The inhibition is comparable for Z-AlaAla-CK and Z-Ala-Phe-ZK, but is especially poor for the shorter analogue Z-Phe-CK where the inhibition was only about 10%. This weak effect is probably due to poor binding of the inhibitor to the substrate-binding site which involves the formation of a pleated sheet structure between the substrate (inhibitor) and two strands of proteinase K and is most favorable for dipeptides or even longer peptides. The influence of methanol on the activity of proteinase K has been studied because we tried to use methanol as a cryosolvent in low temperature crystallographic studies of the enzyme. In assays for enzyme action K m increases with the addition of methanol and, in contrast, the Vma~ are identical in aqueous solution and in presence of methanol (Fig. 5). This result clearly indicates the existence of classical competitive inhibition. Considering the general enzymatic mechanism of serine proteinases with the formation of the acyl enzyme in the first, and the hydrolysis of the acyl enzyme in the second step of catalysis one could assume that methanol competes with water in the second step with the interesting consequence of a trapped transition state complex in presence of methanol even at room temperature. However, according to the ping-pong mechanism proposed for the enzymatic mechanism of serine proteinases (Cleland, 1970) a competitor of the second substrate water would exhibit a non-competitive type of inhibition toward the first substrate Suc-(Ala)a-pNA. Obviously, the formation of the enzyme-substrate complex is affected by methanol which successfully
182
/~sn161~~
~
Fig. 7. Diagram of the active site region in proteinase K, based on the 1.5 ,A resolution X-ray analysis. Side-chains of the active triad, Asp-39-His-69-Ser-224, are drawn solid, and hydrogen bonds by dashed lines. Water molecules are designated OW, the more important water molecules OW328 ( = OW *) and OW335 ( = O W * * ) are filled. We are grateful to Dr. Betzel for permission to reproduce this figure from Ref. 4.
competes for the substrate-binding site because it is in large molecular excess (methanol/Suc(Ala)3-pNA is 4000:1 for 10% methanol).Fig. 4 illustrates the activity of proteinase K as a function of temperature in absence and in presence of 20% methanol. The effect of methanol is negligible at 4%, but it then continuously decreases the activity to about 50% at the temperature optimum, 37 o C. The maximum activity of proteinase K is in the range 37-55 o C. At 70°C, more than 50% of the maximum activity is still retained, indicating a thermal stability in the range as observed for wildtype subtilisin 9. The influence of methanol on the activity of proteinase K can be discussed on the basis of the structure of the active site, which is known to atomic detail from the X-ray analysis. As shown in Fig. 7, the catalytic triad, Asp-38-His-69-Ser224, is in hydrogen bonding contact with water molecules, and several other water molecules hydrogen are bound to the substrate recognition
region (that begins at Ser-132, the beginning of the second strand of the recognition region, Gly-100, is located on the right above Asp-39, not shown in Fig. 7). We can put forward the hypothesis that it is the replacement of water molecules by methanol which competes with substrate binding, thereby increasing the K m. It could be that (i) some or all water molecules in the active site are replaced and (ii) that those water molecules probably involved in the catalytic mechanism are replaced. These are OW335 which is located in the oxyanion hole and OW328 which keeps Asp-39 in the correct conformation so that the Asp 39---His-69 hydrogen bond can form. If these water molecules are replaced by methanol, direct interference with the substrate binding can be anticipated. This holds especially for OW335 whose substitution with methanol would reduce or inhibit binding of the tetrahedral transition intermediate which requires this site for charge delocalization. We could also assume (iii) that methanol binds by hydrogen bonding to Ser-224 and by hydrophobic interactions to the imidazole of His-69, thereby impeding proper binding of the substrate molecules.
Acknowledgement We are grateful to Dr. Burgert, Institut fiar Organische Chemie der Freien Universit~it Berlin, and to Prof. K.-D. Jany, Institut fiir Organische Chemie und Biochemie der Universitat Darmstadt, for providing samples of peptide chloromethyl ketone inhibitors.
References 1 Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H.D. and Land, H. (1974) Eur. J. Biochem. 47, 91-97. 2 PRhler, A., Banerjee, A., Dattagupta, J.K., Fujiwara, T., Lindner, K., Pal, G.P., Suck, D., Weber, G. and Saenger, W. (1984) EMBO J. 3, 1311-1313. 3 Jany, K.D., Lederer, G. and Mayer, B. (1986) FEBS Lett. 199, 139-144. 4 Betzel, C. (1986) Ph.D. thesis, Freie Universit~it Berlin. 5 Laemmli, U.K. (1970) Nature 227, 680-685. 6 Kraus, E., Kiltz, H.-H. and Femfert, U. (1976) HoppeSeyler's Z. Physiol. Chem. 357, 233-237. 7 BrSmme, D., Peters, K., Fink, S. and Fittkau, S. (1986) Arch. Biochem. Biophys. 244, 2, 439-446. 8 Shaw, E. (1970) The Enzymes (Boyer, P., ed.), 3rd Edn., p. 91, Academic Press, New York. 9 Bryan, P.N., Rollence, M.L., Pantoliano, M.W., Wood, J., Finzel, B.C., Gilliland, G.L., Howard, A.J. and Poulos, T.L. (1986) Proteins 1, 4, 326-334.