1325
Langmuir 1992,8, 1325-1329
Subtilisin BPN’: Activity on an Immobilized Substrate P. F. Brode, III,*and D. S. Rauch Miami Valley Laboratories, The Procter & Gamble Company, Cincinnati, Ohio 45239-8707 Received September 18,1991. I n Final Form: March 9, 1992
An immobilized substrate for serine proteases has been prepared, allowing for the simultaneous measurement of enzyme surface activity and hydrolytic activity a t the interface. Subtilisin BPN’ and a single site-directed variant (Y217L, native tyrosine 21’7 changed to leucine) adsorb onto the peptidecoated surface, hydrolyze the peptide, and release it into solution. Adsorption isotherms were measured for BPN’ on the immobilized-substrate surface before and after hydrolysis. This enzyme/immobilizedsubstrate system has been modeled via the Michaelis-Menten equation and thereby provides kinetic parameters for comparison to the enzyme kinetic values reported for the same substrate free in solution. The overall enzyme catalytic efficiency is decreased when the substrate is immobilized. Comparing native BPN’with Y217L, which is catalytically more efficient (1.7X) on a soluble substrate, shows that the variant maintains superior efficiency (1.3X) on the insoluble form of the substrate. Finally, by expressing the data in terms of molecules per area, we are able to show that the hydrolysis rate for the surface-bound substrate increases as the enzyme surface concentration increases.
Introduction Protein adsorption at the solid/liquid interface has been studied for a variety of surface/protein These studies have provided considerable insight into the interaction of naturally occurring proteins with inert solid surfaces. For one class of proteins, enzymes, adsorption may be accompanied by hydrolytic activity on the surface (e.g., lipases? cellulases,6 proteases,’ and amylasess). Among the many examples of interactions between enzymes and insoluble substrates are fabric soil removal in detergency, wood pulp milling in the paper industry, and waste composting in modern efforts t o preserve the environment. Therefore, a full understanding of enzyme interactions with these surfaces requires knowledge of both enzyme adsorption and hydrolysis at the interface. Paralleling the advances in the field of protein adsorption have been many successes using recombinant DNA techniques to produce proteins, specifically proteases, displaying altered enzyme properties.+l3 Soluble synthetic substrates with an attached chromophore are commonly
Materials and Methods
Buffer and Enzyme Solutions. Subtilisin BPN’ (27 534 g/mol) is a serine protease isolated from Bacillus amyloliquejaciens. To produce the variant enzyme Y217L, mutagenesis reactions are performed in Escherichia coli. Native BPN’ and the variant are expressed by a protease-deficientBacillus subtilis strain (supplied by Genencor) during the fermentation process. The following protein purification steps were then applied to the fermentation broth ultrafiltration, ethanol precipitation, DEAE ((diethy1amino)ethyl)Tris Acryl anionic exchange (batch method), SP (sulfopropyl) Tris Acryl cationic exchange (column method), and concentration with an Amicon stirred cell. Enzyme purity was determined to be 295% via high-pressure liquid chromatography (HPLC) and SDS-page (sodium dodecyl sulfatepolyacrylamidegel electrophoresis). The purified enzyme was stored in 5050 propylene glycol/O.Ol M MES buffer (2-(Nmorpho1ino)ethanesulfonic acid) and 0.01 M CaClz adjusted to pH 5.5 with NaOH. The absolute enzyme concentration and the resultant conversion factor (0.0016 (mg min)/(mL AAllo)) for the pNA spectrophotometric assay were determined via a burst titration method.14 This technique employs TCI (N-trans-cinused to quantitate the hydrolytic activity of these variants. namoylimidazole purchased from Sigma) as the titrant. A t the time of use, the enzyme stock solution is eluted through We have investigated the interactions of subtilisin BPN’ a PD-10 (Sephadex-G25) size exclusion column, obtained from wild-type (WT) and the variant (tyrosine 217 changed to Pharmacia, to remove the propylene glycol and exchange the leucine, Y 217L) with a surface containing an immobilized MES buffer for Tris buffer (0.1 M Tris(hydroxymethy1)substrate. Preparation of the immobilized-substrate suraminomethane and 0.01 M CaClz adjusted to pH 8.6 with HC1). face has allowed us to simultaneously study the enzyme Experiments were carried out in the pH 8.6 Tris buffer to match adsorption to, and hydrolysis of, the surface. BPN’s isoelectricpoint and pH maximum for hydrolytic activity. pNA Assay. The pNA assay16 is used to determine the active (1) Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986, 79, 1-63. enzyme concentration by measuring the rate of color production (2) Norde, W. Adu. Collid Interface Sci. 1986, 25, 267-340. (3) Andrade, J. D. Surface and Interfacial Aspects of Biomedical as the enzyme hydrolyzessAAF’F-pNA (succinyl-alanine-alaninePolymers; Plenum Press: New York, 1985; Vol. 2. proline-phenylalanine-p-nitroanilide purchased from Bachem (4) Proteins at Interfaces; Brash, J. L., Horbett, T. A,, Eds.; ACS Inc.). As p-nitroaniline is released in the hydrolysis reaction, Sympoeium Series 343; American Chemical Society: Washington, DC, the yellow color produced is measured at a wavelength of 410 nm 1987. on a Beckman DU-70 spectrophotometer. The pNA assay is (5) Blow, D. Nature 1991,351,444-445. also used in conjunction with absorbance readings taken at 280 Ooshima, H.; Burns, D. S. Appl. Biochem. Bio(6) Converse, A. 0.; technol. 1990,24/25,67-73. nm, yielding total protein concentration,to calculate the enzyme (7) Brode, P. F., 111; Erwin, C. R.; Rauch, D. S.; Armpriester, J. M.; percent activity. Rubingh, D. N. J. Cell. Biochem. 1991, Suppl. 15G, 205 (full publication Preparation and Characterization of CPGsAAPF-pNA in preparation). (8)Henie,Y.I.;Yaron,T.;Lamed,R.;Rishpon,J.;Sahar,E.;Katchalski-Substrate. Aminopropyl controlled pore glass (CPG) having a Darticle size of 125-177 um and a mean Dore diameter of 486 A Katzir, E. Biopolymers 1988,27, 123-138. (9) Wells, J. A.; Estell, D. A. TIES 1988,13, 291-297. i&3.5% pore distribution) was obtained-from CPG Inc. It was (10) Erwin, C. R.; Barnett, B. L.; Oliver, J. D.; Sullivan, J. F. Protein selected as the solid support for the substrate specifically to Eng. 1990,4,87-97. achieve a reasonably high surface area which was accessible to (11) Pantoliano, M. W.; Whitlow, M.; Wood, J. F.; Rollence, M. L.; Finzel, B. C.; Gilliand, G. L.; Poulos, T. L.; Bryan, P. N. Biochemistry
1988,27, 8311-8317. (12) Wells, J. A.; Powers, D. B.; Bott, R. R.; Graycar, T. P.; Estell, D. A. R o c . Natl. Acad. Sci. U S A . 1987, 84, 1219-1223. (13) Russell, A. J.; Fersht, A. R. Nature 1987,328,496-500.
(14) Schonbaum, G. R.; Zerner, B.; Bender, M. L. J. Biol. Chem. 1961, 236, 2930-2935. (15) DelMar, E. G.; Largman, C.; Brodrick, J. W.; Geokaa,M. C . Anal. Biochem. 1979,99, 316-320.
0743-746319212408-1325$03.00/0 0 1992 American Chemical Society
Erode and Rauch
1326 Longnuir, Vol. 8,No. 5, 1992 Scheme I. Covalent Coupling of sAAPF-pNA to CPG
R Rl-N=C=N-Rz
+
R,-c-oH
( E W
1
+
Rl--NT=N-R?
R,NHz
R3-C-0
-
-
Y Rl--N?=N--Rz R C-0 3-0
RY
R,-C--F(R,
Y
+
RY
Rj--N-C--N--R,
d
0 -(CH,),-C-AAPF-pNA
Ri: - - C H , - C H ,
R3:
Rz: -(CH,),
Ra: -(CHz),
-N-(CH3),H'Cl.
-CPG
enzyme. The sAAPF-pNA used in the soluble pNA assay is covalently coupled to the CPG using EDC (l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride purchased from Sigma) as a coupling agent,16and DMSO (dimethyl sulfoxide) as the solvent. A modified pNA assay is used to monitor the completeness of the reaction, shown in Scheme I. The excess solvent is removed from the reaction flask, and CPGsAAPFpNA is transferred to a column for DMSO and Milli-Q water rinsing. CPGsAAPF-pNA is dried at an oven temperature of 65-75 "C with a N2 purge, and stored in a desiccator. A specificsurface area of 50m2/gwas measured on the prepared CPGsAAPF-pNA using the BET (Brunauer-Emmett-Teller) N2 adsorption technique utilized by the Quantasorb Jr. (Quantachrome). CPG Inc. reports a value of 50.0 m2/g for the unmodified CPG, suggesting that the processes involved in the addition of the sAAPF-pNA do not damage the porous structure. The number of available molecules of sAAPF-pNA/pm2 of CPG surface is determined by hydrolyzing the pNA from the surface with an enzyme solution. The CPG:sAAPF-pNA is mixed with successive aliquots of the enzyme solution until no further change is seen in the amount of pNA detected by the 410-nm absorbance of the supernatant. The CPG surface was found to have 62000 f 7000 molecules of pNA/pm2 accessible to the enzyme. Enzyme Adsorption on, and Surface Hydrolysis of, CPGBound Peptide. Tris buffer is added to CPG:sAAPF-pNA in an extended-neck round-bottom flask, and a vacuum is pulled on the flask to degas the pores of the CPGsAAPF-pNA. The flask is contained in a temperature-controlled water bath at 25 "C, and a small volume of an enzyme stock solution is added to the flask to bring the total volume to 30 mL. A wrist-action shaker is used for mixing to avoid damage to the CPG which would result from stirring. At various time intervals the shaker is stopped and the CPG.sAAPF-pNA is allowed to settle (30 s) so that the solution can be sampled. Samples are taken over a period of 90min to ensure that hydrolysis has leveled off. Results of a control experiment run without CPGpNA show no significant loss of enzyme as a result of the method of mixing. Control experimenta with unmodified CPG, providing surface for enzyme adsorption but no substrate to hydrolyze, were also run. Enzyme adsorption by this unmodified surface is about 20% of the enzyme adsorption seen on the peptide-covered surface. An equilibrium level of adsorption on the unmodified surface is achieved within 5 min and is maintained, unlike adsorption on the peptide covered surface, to be discussed later. Samples are analyzed to determine the amount of enzyme adsorbed on the CPGpNA and the amount of pNA hydrolyzed by the enzyme. These are two separate measurements. Adsorption measurements are made first, to minimize the time in which autolysis of the enzyme can occur. The pNA assay is used to measure both the initial enzyme concentration and the enzyme concentration at each sample time. The difference in these concentrations gives the amount adsorbed. By keeping a running total of the mass of BPN' removed from the flask for assaying, and the mass of BPN' remaining in the flask at any given time point in the experiment, we calculate the total amount of BPN' adsorbed. The total micrograms of BPN' adsorbed is then converted to molecules of BPN'adsorbed per square micrometer to be plotted as a function of time. (16) Yamada, H.; Imoto, T.; Fujita, K.; Okazaki, K.; Motomura, M. BiochemLetry 1981, 20, 4838-4842.
a
0
10
20
30
w
-
40
I
0
50
60
Time (minutes)
Figure 1. WT BPN' (0.57 ppm) hydrolysis of (top panel) and adsorption on (bottom panel) 0.1 g of immobilized peptide substrate CPG:sAAPF-pNA.
The hydrolyzed pNA is measured by reading the absorbance of an aliquot of the sample at 410 nm. From this absorbance the total amount of pNA sampled and the amount remaining in the flask are calculated for each time point. These are added to give the total amount of pNA hydrolyzed. A correction which subtracts the amount of pNA that undergoes base hydrolysis in Tris buffer when no enzyme is present is applied to the total micrograms of pNA. The corrected total micrograms of pNA hydrolyzed is then converted to molecules of pNA hydrolyzed per square micrometer to be plotted as a function of time.
Results and Discussion Adsorption on, and Hydrolysis of, CPG:sAAPFDNA. At 25 "C in a 0.1M pH 8.6 Tris buffer, with 10 mM CaClz, we measure a half-life for subtilisin BPN' (0.5 pg/ mL) of over 3 months. We chose these stabilizing conditions t o minimize autolysis while we measured the simultaneous enzyme adsorption on, and hydrolysis of, the surface-bound peptide. T h e two-panel plot of Figure 1 is a typical data set using 0.57 pglmL BPN' and 0.1 g of the CPG:sAAPF-pNA. The lower panel shows the adsorption as a function of time. An adsorption maximum is reached in approximately8 min. Actual adsorption equilibrium for the CPG:sAAPF-pNA surface is shifted, because while in t h e process of reaching an adsorption maximum, the surface concentration of active enzyme begins to decrease. As the enzyme hydrolyzes the peptide surface, a more hydrophilic surface is created in the form of charged carboxy groups. This high energy surface adsorbs less BPN' than the lower energy unhydrolyzed surface. A new equilibrium is continually established at the changing interface, a n d the amount of BPN' adsorbed approaches the value measured for the CPG surface without peptide. In earlier studies of adsorption on a nonporous quartz surface without an immobilized substrate, adsorption equilibrium for a 100 ppm BPN' solution was achieved in 10
Langmuir, Vol. 8, No. 5, 1992 1327
Subtilisin BPN' For this solution, 175times more dilute, equilibrated with the CPGsAAPF-pNA surface, a much longer time (8 min) is required to reach the adsorption maximum. Using the equation of Langmuir and Schaeferls for the rate of transport of molecules to the surface from a wellmixed solution, we can estimate the time ( T ) to achieve the measured level of adsorption (r).For diffusion-ratelimited adsorption, where depletion of the enzyme molecules in the bulk solution is negligible and back-diffusion is neglected, the flux rate is s.17
0
dnldt = Dc/Gx Then
1
I t
0
10
/
1
/
20
40
30
50
60
Time (minutes)
= F/(dn/dt) = r6x/Dc where D (9.8 X lo-' cm2/s) is the diffusion coefficient calculated on the basis of the measured hydrodynamic radius for BPN' (25 A), c (mol/cm3) is the bulk concencm) is the estimated quiescent tration, and 6x (2 X layer thickness. Comparison of the calculated T (8 s) to the actual time to acheive the adsorption maximum (8 min) provides some assurance that the adsorption is not diffusion controlled. Enzyme transport in the pores is an additional consideration because the pores account for a significant portion of the surface area. Using the equation of Brenner and Gaydoslg T
D,,,,/D = 1+ '/J In X - 1.54X for small values of A, where X is the ratio of enzyme molecular radius to pore radius, we calculated a diffusion coefficient for transport in the pores (Dpore)of 5.3 X cm2/s (pore radius for peptide-modified CPG estimated at 220 A). From DVre a characteristic time (t) can be calculated for transport in the pores20 t = fi2/DpOr, where e is the CPG voidage (0.73) and R (cm) is the CPG average particle radius. The value calculated for t is 77 s which suggests that even when transport in the pores is included, the adsorption measured at this enzyme concentration is not transport controlled. The lag time for complete adsorption is paralleled by a lag in the initial rate of hydrolysis of the surface-bound peptide, which is plotted in the upper panel of Figure 1. Followingthe lag, a constant hydrolysis rate is maintained for about 20 min until decreasing substrate concentration slows down the turnover. By 90 min the curve has reached a plateau. Adsorption on the peptide surface is reported as the adsorption maximum, and the corresponding hydrolysis rate is obtained from a least-squares fit of the linear region of the hydrolysis plot. Adsorption and Hydrolysis as a Function of BPN' Concentration. By fixing the amount of substrate at 0.1 g and varying the enzyme concentration, we were able to measure the adsorption isotherm for BPN' on the C P G peptide surface. Figure 2 shows four examples from the series of adsorption (lower panel) and hydrolysis (upper panel) plots for this experiment. As the initial enzyme concentration increasesthe whole adsorption vs time c w e shifts to a higher level of adsorption, and the rate of hy(17)Story,G.M.;Rauch,D.S.;Brode,P.F.,III;Marcott,C. InFourier Transform Infrsred Spectroacopy in Colloid and Interface Science; Scheuing, D. R., Ed.; ACS Symposium Series 447;American Chemical Society: Washington, DC, 1991;Chapter 13. (18)Davies, J. T.;Rideal, E. K. Interfacial Phenomenu; Academic: New York, 1961; p 167. (19)Malone, D. M.; Anderson, J. L. Chem. Eng. Sci. 1978,33,14291440. (20)Do, D. D. Ind. Eng. Chem. Fundam. 1986,25,321-325.
-
I
?
4)
20
30 40 Time (minutes)
10
0
60
50
Figure 2. Hydrolysis of, and adsorption on, 0.1 g of immobilized peptide substrate CPG:sAAPF-pNA using various concentrations of WT BPN' (+, 0.57; @, 1.08;V,2.14; B, 3.38 ppm). Hydrolysis rates are expressed as molecules/(rm2min).
% j 3150
or
0.0
'
'
0.5
'
'
1.0
'
'
1.5
'
'
2.0
'
'
2.5
'
'
3.0
Bulk Equilibrium Concentration (ppm)
Figure 3. Adsorption isotherms for WT BPN' on CPGsAAPFpNA (a) and the same surface after appreciable peptide hydrolysis (0).
drolysis (molecules/(pm2 min)) shows a corresponding increase. In addition, the shape of the adsorption c w e s changes dramatically. At higher enzyme concentrations the time required to reach the adsorption maximum decreases, and the rate of enzyme desorption during hydrolysis increases. The difference between the amount of adsorption on the peptide surface and the adsorption on the hydrolyzed surface becomes greater at the higher BPN' concentrations. Adsorption isotherms are shown in Figure 3 comparing the adsorption for BPN' on the fresh CPG:sAAPF-pNA surface and the CPG after significant hydrolysis of the peptide. At the lower bulk equilibrium concentrations the affinity for the surfaces is much greater than at the intermediate concentrations, as indicated by the sharp rise and then leveling off of adsorption. The steep rise at the lowest enzyme concentrations, especially on the peptide-coated surface, may result from high-affinity specific binding between the enzyme and the surface. Adsorption isotherms for BPN' on hydrophilic and hydrophobic
Brode and Rauch
1328 Langmuir, Vol. 8, No.5,1992
Table I. WT BPN’ and Y217L Kinetic Parameters for Immobilized and S o l u b l W sAAPF-DNA substrate WT immobilized soluble Y217L immobilized soluble
KM,M (6.8 & 1.4) X 1Oa 1.4 X lo-’ (1.55 f 0.04) X lo-‘ 4.7 x 10-4
k,JKM, kat, 8-l
*
e-’
M-1
0.94 0.18 13 900 50 357 OOO 2.8 0.07 18 300 280 596 OOO A
0
10
20
30 40 Time (minutes)
50
60
80
I
1
h
60 v
za 0
40
n
LI/
6
4
a
1-
x
v
1
20
0
10
30 40 Time (minutes)
20
50
60
Figure 4. Mass of sAAPF-pNA hydrolyzed and mass of WT BPN’ adsorbed using different amountsof sAAPF-pNAsubstrate (A, 0.60; m, 0.30;V, 0.18; 0, 0.13; +,0.10 g). Initial WT BPN’ concentration was held constant at 2.2 ppm.
(methylated) quartz show that at low bulk concentrations the hydrophobic surface adsorbs a t least an order of magnitude more than the hydrophilic surface.” The adsorption isotherm for WT on CPG:sAAPF-pNA is a close match to the isotherm for the hydrophilic quartz, both displaying a very low BPN’ surface density. Comparing the 50-100 molecules/pm2 of Figure 3 with the 50 O00 molecules/pm2 of a close-packed monolayer gives an appreciation of the large space available between each adsorbed BPN’ molecule. Enzyme Kinetic Parameters on an Immobilized Substrate. Although the Michaelis-Menten mechanism is not appropriately applied to the enzyme/immobilizedsubstrate system described here, we wanted to see if the Michaelis-Menten equation could be used as a suitable fit to the data.21 Via the classic approach we measured a series of adsorption/hydrolysis sets in which the BPN’ concentration was held constant at 2.2 pg/mL and the amount of immobilized substrate was varied. These data for WT are presented in the two panels of Figure 4 as mass of BPN’ adsorbed and mass of pNA hydrolyzed vs time. Plotting the data in terms of mass gives the best appreciation of what is occurring in the solution as a function of substrate concentration. As the concentration of immobilized substrate is increased, the absolute amount of active enzyme adsorbed by the solid increases. Likewise, the increased substrate produces an increase in both the initial rate and the absolute amount of hydrolyzed peptide in the solution. When these hydrolysis data are expressed in molar units and plotted via the double-reciprocal (Lineweaver-Burk) form of the Michaelis-Menten equation, linear fits give correlation coefficients of 0.9919 and 0.9999, for WT and Y217L, (21)Fereht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H. Freeman and Co.: New York, 1985;Chaptes 3.
respectively. The estimated standard deviation (SD) for the slope and y-intercept were calculated for these plots. These values were propagated to the kinetic parameters described below and are included in Table I. Using the values from the slope and y-intercept, and the Lineweaver-Burk equation, the kinetic parameters KMand kat are calculated for WT and the variant enzyme on the immobilized sAAPF-pNA. The apparent binding constant (KM) and catalytic rate constant (kat) can be used for comparison to the values obtained on the same ‘mobilized” substrate in s o l ~ t i o (Table n ~ ~ ~I).~ ~ Subtilisin BPN’ has a turnover rate, kcat,for the soluble substrate which is 50 times faster than on the same substrate immobilized on the glass surface. Alternately the K M for the immobilized substrate is about half the value for the soluble substrate. Therefore, the catalytic efficiency (k,,JKM) for the immobilized substrate is reduced by a factor of 25 over the value for soluble pNA. The significanceof the k,J& is that it relates the reaction rate to the free substrate concentration and the free enzyme concentration. It is an apparent second-order rate constant,because the k,J& value is a measure of the slowest second-order k in the forward reaction path. Only in the case where the rate-determining step is the encounter of enzyme and substrate does it represent the true rate constant. Hence, the catalytic efficiency sets a lower limit for the association of free enzyme and substrate.21 A similar comparison can be made for the kinetic parameters of the Y217L variant. This variant of BPN’ has an increased activity on soluble substrates23 (1.7X) and likewise is more active on the immobilized sAAPFpNA. Y217L’s turnover rate for the soluble sAAPF-pNA is almost 100times faster than its rate on the immobilized substrate. The K M for the insoluble substrate is about one-third the value for the soluble form of sAAPF-pNA. This translates to a catalytic efficiency which is reduced by a factor of 33 when the substrate is bound to a surface. Y217L is still superior to WT on insoluble substrates (1.3X); however, it is apparently affected more by the substrate being bound to a surface. Although these values are useful for comparison, we have not attempted to use them to propose a viable mechanism due to the complexity of the processes involved in turnover of the immobilized substrate (adsorption, desorption, surface diffusion, and orientation of the adsorbed BPN’). Hydrolysis and Adsorption from a Surface-Area Perspective. Another way to evaluate the results from the variable substrate (fixed [El,) series (Figure 4) is to use the measured surface area, and calculate adsorption and hydrolysis in terms of molecules per area (BPN’ adsorbed or pNA hydrolyzed per unit surface area). In this way what is happening at the interface is highlighted, as opposed to the preceding discussion of total enzyme and substrate in the solution. Comparing these two ways of representing the data shows that adsorption and hydrolysis correlate with substrate concentrations,but in opposite (22)Estell,D.A.;Graycar, T. P.; Miller, J. V.; Powers, D. B.; Burnier, J. P.;Ng,P. G.; Wells, J. A. Science 1986,233,659-663. (23)Wells, J. A.;Cunningham, B. C.; Graycar, T. P.; Estell,D. A. h o c . Natl. Acad. Sci. U.S.A. 1987,84, 5167-5171.
Subtilisin BPN’
Langmuir, Vol. 8, No. 5, 1992 1329
Table 11. Adsorption to, and Hydrolysis of, CPG:sAAPF-pNA Expressed in Terms of Absolute Concentration and Molecules per Area CPG: sAAPFpNA, mg 100.0 125.3 180.0 301.3 602.5
BPN’ adsorption, C I g
22.2 27.3 36.8 53.5 63.2
pNA hydrolysis, pg min-1 3.61 4.48 6.05 8.54 9.84
BPN‘ PNA adsorption, hydrolysis, molecules molecules pm-2 pm-2 min-1 91.3 3150 95.2 3120 2930 89.5 17.7 2470 45.9 1420
directions for absolute and per-area values (Table 11). As the amount of insoluble substrate increases, the total surface area must increase, resulting in a lower surface concentration of BPN’. With this decrease in surface concentration of BPN’ comes a resultant decrease in the rate of peptide hydrolyzed per unit area. Although the absolute amount of enzyme adsorbed and substrate hydrolyzed increases with increasing sAAPF-pNA, the amount of both adsorbed enzyme and hydrolyzed substrate per unit area decreases.
Summary We have been able to prepare an immobilized substrate for subtilisin BPN’ which allows us to measure the surface activity of the enzyme and simultaneously observe the hydrolytic activity at the interface. Subtilisin BPN’ adsorbs onto the peptide-coated surface, hydrolyzes the peptide, and releases it into solution. As the enzyme pro-
gressively hydrolyzes the peptide from the surface, the adsorption equilibrium is shifted toward desorption back into the bulk solution. These investigations on subtilisin BPN’ conclude that surface coverage and affinity are greater on the peptide-covered surface than on the aminopropyl controlled pore glass surface, which may be the result of specific binding between the enzyme and the immobilized substrate. The enzyme/immobilized-substrate system can be modeled via the Michaelis-Menten equation and thereby provides values for comparison to the enzyme kinetics on the same substrate free in solution. The overall enzyme catalytic efficiency is decreased when the substrate is immobilized. Comparisonswith the more catalytically efficient (on a soluble substrate) Y217L variant show that it remains superior to WT on the insoluble form of the substrate. By expressing the data in terms of molecules per area we are able to show that the hydrolysis rate for the surface-bound substrate increases as the enzyme surface concentration increases.
Acknowledgment. Our thanks go to Ken Williams and Chris Erwin for the purification of the wild-type subtilisin BPN’, Rick Fowler and Jim Thompson for consultation on binding the peptide to CPG,Donn Rubingh, John Sullivan, Bobby Barnett, and Dave Melik for many useful discussions and helpful suggestions over the course of the work, and Judy Richardson for preparation of the paper. Registry No. SAAPF-p-NA,70967-97-4; subtilisin, 9014-011; tyrosine, 60-18-4.
