kinetic analysis of the mechanism for subtilisin in essentially anhydrous organic solvents

Kinetic analysis of the mechanism for subtilisin in essentially anhydrous organic solvents

Sudipta Chatterjee and Alan J. Russell

Center for Biotechnology and Bioengineering and Department o f Chemical Engineering, University o f Pittsburgh, Pittsburgh, Pennsylvania

Steady-state kinetic analysis has been used to confirm the catalytic mechanism of lyophilized subtilisin suspended in a variety of organic solvents. Specifically, this article demonstrates that partial reactions can occur between subtilisin and ester substrates in organic solvents. Partitioning of common intermediates between competing acceptors at a constant ratio of products has also been described. The decomposition of a common intermediate formed from different substrates at the same rate is also further evidence of an acyl-enzyme mechanism for subtilisin suspended in anhydrous solvents. Parti- tioning of a common intermediate to give two products at a constant total rate, and saturation kinetics at varying substrate concentrations, complete a kinetic investigation of the enzyme mechanism. All the data generated support the formation of a stable acyl enzyme during the transesterification reaction catalyzed by subtilisin in the solvents used.

Keywords: Enzymes; organic solvents; mechanism; subtilisin; microscopic rate constants; active site titration

Introduction

Elucidation of the mechanism of any enzymatic reaction involves the establishment of the sequence of the elementary reaction steps, characterizat ion of the various complexes and intermediates formed in the pathway, and determination of their rates of interconver- sion. Whereas detection, isolation, and structural characterizat ion of intermediates are considered as di- rect proof of the route or pathway for a reaction, it is often not possible to dissect the catalytic machinery to this degree. An intermediate can also be considered to belong to a pathway if kinetic studies demonstrate consis tency with a proposed hypothesis. Indeed, the relationship between rate and reaction conditions is usually the main criterion for assignment ofbiocatalyt ic reaction pathways.

A useful model enzyme for kinetic studies is the serine protease subtilisin. Over the last 50 years, the kinetics of subtilisin-catalyzed ester and amide hydro-

Address reprint requests to Dr. Russell at 1235 Benedum Hall, De- partment of Chemical Engineering, University of Pittsburgh, Pitts- burgh, PA 15261 The current address of Sudipta Chatterjee is Michigan Biotechnology Institute, Lansing, MI 48909 Received 24 February 1993; revised 27 April 1993

lysis have been studied with many substrates, t-3 In water, peptide and synthetic ester substrates are hy- drolyzed by serine proteases via the acyl-enzyme mechanism (for a detailed description, see reference 9). The enzyme and substrate first associate noncova- lently to form the enzyme-subs t ra t e complex. This is followed by attack of the hydroxyl group of the active- site serine residue upon the carbonyl of the substrate, forming the tetrahedral intermediate in which the oxya- nion of the substrate is stabilized by the backbone chain of the protein. The collapse of this intermediate leads to the release of the amine or alcohol and to the formation of the covalent acyl-enzyme intermediate. After hydrolysis resulting from the attack of water, the enzyme-p roduc t complex is formed, which then col- lapses to release free enzyme and product. More re- cently, research directed at understanding how a solvent can be used to control the activity of subtilisin has led to kinetic studies of subtilisin in predominantly nonaqueous media. 4 In such an environment the enzyme is insoluble, and subtilisin-catalyzed transesterification replaces ester hydrolysis as the reaction of choice. The importance of these kinetic studies should not be underestimated. Comparison of enzyme activi- ties in different solvents, and the development of pre- dictive models relating enzyme activity to solvent structure, depends on a detailed understanding of the effect of solvent on mechanism and the rates of individ-

1022 Enzyme Microb. Technol., 1993, vol. 15, December © 1993 Butterworth-Heinemann

Subtilisin in organic solvents: S. Chatterjee and A. J. Russell

ual steps during catalysis. Mechanistic analyses of subtilisin-catalyzed transesterification reactions in organic media, to date, include investigations of Hammett rela- tionships, 5 primary kinetic isotopic effects, 6 chemical modification of active site residues,7 and determination of individual rate constants for a hypothesized mechanism. 8 All these studies imply that the classical acyl- enzyme mechanism for ester hydrolysis operates during transesterification. There are a number of classical kinetic approaches for proof of a ping-pong mechanism which have yet to be presented in the literature. 9,1° In this article we demonstrate that kinetic tests used to prove that subtilisin-catalyzed ester hydrolysis in water proceeds via an acyl-enzyme intermediate can be used to substantiate an identical mechanism for subtilisin- catalyzed transesterification in an organic solvent.

Pre-steady-state kinetics are often used to investi- gate the rates of individual reactions within a pathway. ILl2 Unfortunately, the heterogeneous nature of enzyme-catalyzed processes in organic media severely limits the application of these delicate techniques. Thus, one must turn to steady-state techniques for substantiation of the acyl-enzyme mechanism by kinetic experimentation in predominantly nonaqueous environments. The following kinetic tests, which are described in Figure 1, must hold for the acyl-enzyme mechanism to be formally substantiated.l°

1. Demonstration o f partial reactions: If transesterification reactions catalyzed by subtilisin in organic media do indeed follow the ping-pong (acyl-enzyme) mechanism, then the overall reaction should proceed through two half-reactions. Hence, it should be possible to carry out an acylation reaction between the first substrate (for example, an ester) and the enzyme, even in the absence of the second substrate (alcohol). For- mation of the first product should then confirm the first step in the two-step ping-pong mechanism.

2. Partitioning o f common intermediates between competing acceptors at a constant ratio o f products: For the acyl-enzyme mechanism, the common intermediate, RCO-E, formed from different substrate donor molecules, RCO-X and RCO-Y, should give a constant ratio of products, RCONI and RCON2, at given concentrations of nucleophiles, NI and N2. A constant product ratio can again be considered as evidence of formation of a common intermediate, RCO-E.

3. Decomposi t ion o f a common intermediate formed f rom different substrates at the same rate: If the deacylation of the common intermediate, RCO-E, is the rate-determining step in the overall reaction, then the rate of product, RCON, formation should be the same for substrates that give rise to a common intermediate, provided the concentration of nucleophile, N, remains the same.

4. Partitioning o f a common intermediate to give two products at a constant total rate: When the common intermediate, RCO-E, deacylates to form two products, RCON 1 and RCON2, under steady-state conditions, the sum of the rates of formation of the two products of deacylation should be equal to the rate

Partitioning of common intermediate between competing acceptors at a constant ratio

~COX

or

~COY

+ E

RCON i+ E

RCOE i -

+ ~ RCON + E YorX 2

Partitioning of common intermediate to give two products at a constant total rate

~COX + E

RCOE Y RCONI+ E

+ ~ RCON2+ E X

Decomposition of common intermediate formed from different substrates at the same rates

¢COX

gCOY

N + E ~ RCOE ~ RCON + E

+

YorX

Figure 1 Steady-state kinetic tests for substantiation of a common intermediate. In the above schemes, RCO-X and RCO-Y represent two substrates which differ only in their leaving groups, X and Y respectively. N1 and N 2 represent two different acceptor molecules, and E represents the enzyme

of formation of X (and therefore that of the common intermediate RCO-E) if the acyl enzyme mechanism operates.

5. Saturation kinetics at varying substrate concentrations (see below).

For the acyl-enzyme mechanism, as outlined in Fig- ure 1, turnover number (kcat) , Michaelis constant (Kin),

( cat and the specificity constant \Km/ for the ester sub-

strate may be given by equations (1-3):

k2k3[82]

kcat - k2 + k3182] (1)

Ksk3[S2] Km - k2 + k3182] (2)

Km_Ks kcat k2 (3)

Enzyme Microb. Technol., 1993, vol. 15, December 1023

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where [82] represents the concentration of the second (alcohol) substrate. Equations (1) and (2) may also be written in their reciprocal forms

1 1 1 - - + ( 4 )

kcat k3[$2] k2

1 k 2 l 1 (5)

K m K~k3[S 2] K~

According to equations (4) and (5), 1/kca t and 1/K m both show a linear dependence on [ 1/$2]. However , the specificity constant, kcat/Km, is independent of the concentration of $2. We have discussed previously how the introduction of an added nucleophile ($3) influences the kinetics of deacylation and thereby enables the determination of the rate constants for acylation and deacylation. 8 If a common intermediate exists, as rep- resented by acyl-enzyme formation, then under steady- state conditions the rates of acylation and total deacylation should be equal. Under conditions where the rate- determining step is known (for instance, after having measured the actual rates of acylation and deacylation), this situation could be described by equation (6). Experimentally the left-hand side of equation (6) may be determined from saturation kinetics with one ester and alcohol. The right-hand side of the equation may be obtained from microscopic rate constants determined by the added nucleophile method using one ester and two alcohols:

1 / Kin/formation of P1 \Krn/ format ion of P2

\gm/formation of P3 (6)

This article presents a set of kinetic data that substantiate the hypothesis of a common intermediate (the acyl-enzyme). Given the substantial amount of work already published on the kinetic behavior of subtilisin in organic solvents, along with the determination of its structure in anhydrous environments, it can be as- sumed that the enzyme-catalyzed transesterification proceeds in the same manner as for enzyme-catalyzed hydrolysis. Further, since the overall activity of the enzyme in organic solvents is much reduced, relative to water, yet the same mechanism holds, protein or solvent engineering should be possible to increase ra- tionally the activity of subtilisin in organic solvents.

E x p e r i m e n t a l p r o c e d u r e s

For these studies we examined transesterification reactions between N-protected amino acid esters such as N-aceyl-e- phenylalanine methyl ester (Ac-Phe-OMe), N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt) and N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), and n-propanol (PrOH) and n-butanol (BuOH). All substrates and solvents were of the highest purity commercially available and were purchased from Sigma Chemical Co. (St. Louis, MO).

Enzyme and solvent preparation

Subtilisin was freeze-dried from pH 7.8 phosphate buffer, ionic strength 0.01 M (10 mM CaC12), for 48 h on a Labconco lyophilizer (Model 4451 F). All reactions were carried out in tetrahydrofuran. Tetrahydrofuran was dried by using molecu- lar sieves prior to use. The water content of the solvent was 0.2% by weight as measured by Karl Fisher titration. The lyophilized enzyme powder had a water content of 20% by weight as measured by Karl Fisher titration.

Initial rate determinations

The rates of transesterification reactions were determined at 50 mM concentration of the ester substrates. The concentrations of the two alcohols, propan-l-ol and butan-l-ol, varied from 30 to 60 mM, as required. Typically, 3-4 mg of enzyme powder was weighed out in 4-ml Wheaton vials. A solution of the substrates (ester and alcohol) was then added to the enzyme and the reactions were initiated by sonicating the reaction mixtures for 10-15 s. The vials were then placed inside a shaker/incubator (New Brunswick, G24) at the required temperature of reaction (30 or 43°C) at 325 rev rain -~. At this shaking speed, the reaction was experimentally determined not to be limited by external diffusion.

Reaction rates were determined by following the formation of either the ester or the alcohol using a gas chromatograph (HP 5890 Series II, equipped with an HP1 crosslinked methyl silicone capillary column for ester and a Supelco 60/ 80 Carbopack B/l% SP 1000 packed column for alcohol detection with an FID detector). Temperature ramps were chosen in order to be able to detect both substrate and product within 5 min of injection. At regular intervals, 0.2-p.l reaction samples were injected into the gas chromatograph for product analysis. All reactions were followed for approximately ! h. Initial reaction rates were determined from the slopes of the linear regression plots of product formation versus time, using a linear regression analysis program (Enzfitter by Robin Leatherbarrow, Elsevier, Cambridge). Saturation kinetic studies were performed at a variety of temperatures and 325 rev min -j. All reactions were followed up to 2% conversion. All other conditions were as described above.

Partial reactions were performed at 27°C and 325 rev min between p-nitrophenylbutyrate and 2.3 mg of enzyme powder in hexane, acetonitrile, and dioxane. Reaction rates were determined by following the formation ofp-nitrophenol using a gas chromatograph (HP 5890 Series II, equipped with an HP1 crosslinked methyl silicone capillary column and an FID detector). Temperature ramps were chosen in order to be able to detect both substrate (p-nitrophenyl butyrate) and product (p-nitrophenol) within 5 rain of injection. At regular intervals, 1-/~1 reaction samples were injected into a gas chromatograph (HP 5890 Series II) for product analysis. All reactions were followed up to at least 2% total conversion.

R e s u l t s

Partitioning o f common intermediate between competing acceptors at constant ratio o f products

Table 1 shows the product ratios for deacylation of three esters, Ac-Phe-OMe, Ac-Phe-OEt , and Ac-Tyr- OEt, in the presence of 30 mM propanol and 30 mM butanol at 43 and 325 rev min -1. The two esters of phenylalanine gave similar product ratios for {propanolysis/butanolysis}, indicating that both esters form the

1024 Enzyme Microb. Technol., 1993, vol. 15, December


Table 1 Partit ioning of common intermediate at 43°C and 325 rev min -1

Propyl ester Butyl ester mM mM Propanolysis

Substrate a min -1 × 102 rain -1 × 102 Butanolysis

N-Ac-Phe-OMe 3.92 -+ 0.40 2.12 +- 0.03 1.85 N-Ac-Phe-OEt 4.12 -+ 0.42 2.17 -+ 0.02 1.89 N-Ac-Tyr-OEt 0.98 -+ 0.01 1,52 -+ 0.02 0.65

All results are based on an enzyme concentration of 1 mM a All substrates were maintained at a concentration of 50 mM

alcohol are tabulated in Table 4. Comparing the rates of nonenzymatic and enzymatic reactions in hexane, dioxane, and acetonitrile, we can say that in dioxane and acetonitrile a slow partial reaction (acylation) takes place even in the absence of the alcohol $2:

p-NPB + E ~ p-NP + EA (6)

where p-NPB, E, p-NP, and EA are p-nitrophenylbutyrate, enzyme, p-nitrophenol, and acyl-enzyme, respectively. The rate of acylation, as measured by the formation of p-nitrophenol, is higher in dioxane and acetonitrile than in hexane.

Table 2 Decomposit ion of common intermediate f rom different substrates at 43°C and 325 rev min -1 with propanol (30 ma)

Substrate a Rate b (mM min 1)

N-Ac-Phe-OMe 6.67 x 10 2 N-Ac-Phe-OEt 6.62 × 10 -2

o Both substrates were at a concentration of 50 mM b Rates are based on 1 mM enzyme concentration. Errors are -+10%

same intermediate during transesterification. How- ever, on comparing the ratios for Ac-Phe-OEt and Ac- Tyr-OEt, it becomes evident that dissimilar complexes partition at different ratios. All experiments were performed with the same amounts of enzyme, ester, and alcohol and were carried out over the same length of time.

Decomposition of common intermediate formed from different substrates at the same rate

The rates of deacylation of Ac-Phe-OMe and Ac-Phe- OEt in the presence of 30 mM propanol are shown in Table 2. These studies were carried out at 43°C and 325 rev min -~, where previous studies have shown that deacylation is rate controlling. The rate of formation of propyl ester is the same for both substrates, which implies that both give rise to a common intermediate during transesterification.

Partitioning of common intermediate to give two products at constant total rate

The results shown in Table 3 indicate that at 28°C the rate of formation of the first product, ethanol, does not change upon an increase in the concentrations of nucleophiles from 30 mM propanol and butanol to 60 mM. This indicates that at this temperature acylation is rate determining.

Demonstration of partial reaction as proof of ping-pong mechanism

The results of the partial reaction study between subtilisin and p-nitrophenol butyrate in the absence of an

Saturation kinetics

The most common example of evidence for an acyl- enzyme mechanism is the parallel line plot in which the apparent K m and kca t for the ester substrate are determined at varying concentrations of the alcohol. In Table 5 we present the data for such an experiment, and Figure 2 describes the dependence of specificity constant and turnover number on the concentration of alcohol.

Saturation kinetics can also be used in the presence of an added nucleophile to provide evidence for the formation of an acyl-enzyme intermediate. In Table 6, (kcat~ - - / was determined from saturation kinetic analy- gm//ester

sis using the same method as that used to generate the

data in Table 5. The values for (kent I and \Kin/alcohol 1

kcat) inTable6, however, werecalculatedfrom gm/alcohol 2 microscopic rate constants determined in the presence of an added nucleophile as described previously. 8

Since (kcat) (kcat) + (kcat) 2}' \Km/este r equals {\Km/alcohoi I \Km/alcohol

the expected kinetics for the acyl enzyme mechanism are satisfied.

Discussion

Partitioning of common intermediate between competing acceptors at constant ratio of products

If transesterification reactions with subtilisin follow the acyl-enzyme mechanism, then although Ac-Phe-OMe and Ac-Phe-OEt have different leaving groups (-OMe and -OEt), the same acyl-enzyme intermediate (phenylalanine actyl-enzyme complex) will be formed during reaction with either substrate. Conversely, although Ac-Phe-OEt and Ac-Tyr-OEt have the same leaving group (-OEt), different expected intermediates (Ac- Phe-O-Enz and Ac-Tyr-O-Enz) will be formed during the reaction. If the acyl enzyme can be deacylated by either of two nucleophiles, then identical intermediates will result in the synthesis of fixed concentrations of propyl and butyl ester over a given time. The relative amounts of propanolysis and butanolysis would be ex-


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Table 3 Partitioning of common intermediate from N-Ac-Phe-OEt to give two products at 28°C and 325 rev min -1

Rate of Rate of Rate of Rate of formation of formation of formation of formation of

PrOH BuOH BuOEt PrOEt EtOH PrOEt + BuOET (mM) (mM) (mM min -1) (mM min -1) (mM min -1) (mM min 1)

30 30 2.20 x 10 2 4.46 × 10 _2 8.20 x 10 -2 6.70 x 10 -2 60 60 2.70 x 10 -2 5.12 x 10 2 8.43 × 10 -2 7.82 x 10 -2

All rates are based on 1 mM enzyme concentration. Experimental error is -+13%

Table 4 Partial reaction studies with p-nitrophenol butyrate at 28°C and 325 rev min -1

Solvent

0,01

Hexane CH3CH Dioxane

0.008 Rate

Rate non-enzymatic enzymatic Actual rate of .~ reaction reaction acylation a 0.oo6

(~iMn) (m~n) (min~genz ' ) 0.004

0.030 (-+10%) 0.036 (-+30%) 5.22 X 10 3 ~,B 0,065 (-+12%) 0.095 (--+20%) 2.61 × 10 -2 0.017 (--+10%) 0.048 (-+20%) 2.70 X 10 2 O.0O2

All experiments were performed with 50 mM p-n i t ropheny l bu- ty ra te

1 0

pected to be related to the structure of the particular acyl enzyme. Naturally, uncommon intermediates would deacylate differently and give different ratios of propyl and butyl esters.

Our results (Table 1) show that the transesterification reaction between Ac-Phe-OMe or Ac-Phe-OEt and a propanol/butanol mixture results in the same ratio of propyl to butyl ester product. On the other hand, comparing {propanolysis/butanolysis} for either Ac- Phe-OEt or Ac-Phe-OMe to Ac-Tyr-OEt indicates dissimilar ratios of product. Clearly, Ac-Phe-OEt and Ac- Phe-OMe form the same common intermediate during transesterification with subtilisin in organic media. In water similar partitioning experiments with chymotrypsin have been useful in proving the formation of a common acyl-enzyme complex during hydrolysis. Chymotrypsin-catalyzed hydrolysis of a series of hip-

1 8

I ° , ca, I i . . . . . "[

z /

r ~ i l 1 - -

0.004 0008 0.012

4

r

2

0 0 0 0 016 0.02

1 / [ M e O H ] ( r a M )

-3

Figure 2 Effect of alcohol concentration on the kinetic constants for the subtilisin-catalyzed transesterification of N-acetyI-L- pheny la lan ine ethyl ester by methanol. The zero slope of the line indicates the parallel nature of the individual Lineweaver- Burk plots

purate esters in the presence of hydroxylamine is reported to give a constant product ratio of hydroxamic acid to free acid, proving the formation of a common hippuryl enzyme intermediate in each case.13

The success of this method depends upon the accumulation of a common and stable intermediate. The formation of an acyl-enzyme complex is consistent with the kinetic data. Related work on temperature effects for this reaction over a range of 32 to 60°C have shown that above 37°C deacylation is rate controlling. Hence, a temperature of 43°C was used in these studies

Table 5 Saturation kinetic studies with N-acetyI-L-phenylalanine ethyl ester at 50°C and 325 rev min 1 in tetrahydrofuran

1 1 1 Km

Conc. MeOH [MeOH] Krn kca t kcat (mM) (mM -1) (mM 1) (min) (mM min)

60 1.66 x 10 2 1.92 x 10 -2 6.50 2.95 x 10 3 80 1.25 x 10 2 2.20 x 10 -2 6.67 3.3 x 10 3

100 1.00 x 10 -2 1.27 x 10 2 4.30 2.95 x 10 3 120 8.33 x 10 -3 1.20 × 10 -2 3.65 3.3 x 10 -3

All reactions were in THF and all rates are based on an enzyme concentration of 1 mM. Errors in kca t were -+9%, errors in Km were +28%

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Table 6 Saturation kinetic studies with benzoly-L-alanine methyl ester (BAME) and N-acetyl-L-phenylalanine methyl ester

Km/alcohol 1 +

(kcat) (kcat t (/(cat) (/(cat) Substrate \ Km/ester \ Km/alcohol 1 \ Km/alcohol 2 \ Km/alcohol 2 ( s o l v e n t ) ( m i n -1 M -1) (m in -1 M -1) ( ra in -1 M -1) ( ra in -1 M -1)

Ac- Ph e-O M e a 1.000 0.586 0.445 1.031 (THF)

BAME h 0.765 0.457 0.284 0.741 (THF)

BAME b 0.390 0.217 0.183 0.400 (acetonitrile)

BAME b 0.340 0.171 0.159 0.330 (dioxane)

All rates are based on 1 mM enzyme concentration, a Reactions with Ac-Phe-OMe were at 42°C. b Reactions with BAME were at 30°C

to ensure the accumulation of acyl-enzyme complex. The need for deacylation to be rate limiting is not, however, absolute. Hydrolysis of amides by the acyl- enzyme mechanism is acylation rate controlled, and there is no accumulation of the acyl-enzyme complex. Nevertheless, Fastrez and Fersht =4 have found the product ratio of {transacylation/hydrolysis} to be the same for phenylalanine amide and phenylalanine ester in the presence of different acceptor nucleophiles such as alanine amide and glycinamide. Their results, it is argued, indicate that hydrolysis and aminolysis proceed through the formation of a common intermediate.

Decomposi t ion o f common intermediate f o rmed f rom different substrates at the same rates

The deacylation of a series of substrates that have the same acyl group (but different leaving groups) by the same nucleophile will occur at the same rate, provided the concentration of the nucleophile remains the same in all cases. Transesterification of the methyl and ethyl esters of phenylalanine by 30 mM PrOH at 43°C (where deacylation is rate controlling) gives equivalent rates of formation of propyl ester (Table 2). Once again, the results indicate that deacylation is taking place from a common complex, and in turn point towards the acyl- enzyme mechanism for transesterification. Zerner et al.=5 have determined the rate constants for acylation and deacylation for a chymotrypsin-catalyzed hydrolysis of the amide and the ethyl, methyl, and p-nitrophenol esters of N-acetyl-L-tryptophan by partitioning experiments. They obtained the same value of rate constant for deacylation with all the substrates.

Partitioning o f common intermediate to give two products at constant total rate

We have considered the subtilisin-catalyzed alco- holysis of Ac-Phe-OEt with two different nucleophiles (PrOH and BuOH) at 28°C. At this temperature we hypothesized that acylation was the rate-controlling

step. Steady-state kinetics dictate that the rate of formation of ethanol (the first product) should be equal to rate of formation of the acyl-enzyme complex, which in turn should equal the sums of the rates of formation for the propyl and butyl ester products (products 2 and 3).

The results presented in Table 3 indicate that the rate of formation of EtOH is slightly higher than the sum of the rates of propanolysis and butanolysis. There are two plausible reasons for this behavior. Hydrolysis of the acyl-enzyme complex by the water present in the system could account for the surprising result, but acid formation was not detected by gas chromatogra- phy. Rather, it is more likely that errors due to the determination of EtOH (P0 and ester (Pz and P3) concentrations using two different gas chromatographs account for the behavior. Nevertheless, the constancy of the rates of formation of EtOH, even under changing concentrations of alcohol, indicates that acylation is rate controlling under the conditions of reaction. In other words, the formation of a common intermediate is the rate-controlling step for transesterification reactions with both substrates Ac-Phe-OMe and Ac-Phe-OEt.

Partitioning experiments of the type described above are limited by the degree of product inhibition, the degree of substrate inhibition, the degree of diffusional limitations, the relative rates of acylation and deacylation, and the potential of reaction of acceptor nucleophiles with the enzyme-substrate complex before de- parture of the leaving group. We have also found that the best kinetic results are obtained when all the experiments are performed with the same batch of enzyme, and the concentration of enzyme is constant in all assays. The physical properties of the solvent in which an enzyme is placed exert significant effects on activity and specificity; therefore it is important to compare kinetic experiments performed in a single solvent.

Demonstration o f partial reactions

If transesterification reactions catalyzed by subtilisin in organic media follow the ping-pong mechanism, then

E n z y m e M i c r o b . T e c h n o l . , 1993, vo l . 15, D e c e m b e r 1027

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I 0 - - i i - T i

8 -

J !

2

J

o ~ i i i

0 40 80 120 160 200

Time (min)

Figure 3 Progress curve for the partial reaction between subtilisin and p-nitrophenyl butyrate in dioxane. Details are given in the text

the overall reaction should proceed through two half- reactions (equation 6). Further, an acylation reaction between the first substrate (ester) and the enzyme, even in the absence of the second substrate (alcohol), should result in the formation of the first product. The amount of product produced under these conditions will de- pend on the ability of the enzyme to bind its substrates. In organic solvents the binding of amino-acid ester substrates is decreased relative to water, and it was not possible to observe acylation in the absence of an external nucleophile. However, improving the ester leaving group by utilizing p-nitrophenylbutyrate as the substrate enables the partial reaction to proceed. The results presented in Table 4 demonstrate the formation ofp-nitrophenol (PI) in the absence of an added nucleophile in both acetonitrile and dioxane (in hexane there is no observable acylation). When the same reaction was studied in the presence of EtOH, the formation of ethyl butyrate in addition to p-nitrophenol proves that the overall reaction is indeed transesterification. Of the three solvents used, dioxane (log P = -1.1) and acetonitrile (log P = -0.36) are more hydrophilic than hexane (log P = 3.52). Higher rates of acylation in acetonitrile and dioxane as compared to that in hexane may be explained on the basis of improved binding of substrate to enzyme in more hydrophilic solvents. In water, proof of acyl-enzyme mechanism during hydrolysis by such a partial reaction is not possible, since water is both the nucleophile ($2) and the medium. The partial reaction is of particular utility because it enables the design of an in situ active-site titration method for subtilisin in organic media. A continuous feed of single substrate will eventually lead to complete reaction of all active sites, which upon the subsequent addition of nucleophile will enable an assay of the total ester product, giving the total concentration of enzyme. This method will be described in detail in a future article, although a progress curve for the partial reaction in dioxane is given in Figure 3.

Proof o f hypothesized mechanism by steady- state saturation kinetic studies

We have used two different saturation kinetic techniques to substantiate the mechanism of transesterification. In Table 5, the values of kca t, K m, and Km/kca t for transesterification of Ac-Phe-OEt are given at various concentration of MeOH. The validity of the proposed mechanism is shown by the fit of experimental data to equations (3) and (4) developed on the basis of the acyl- enzyme mechanism (Figure 2). Correlations of this type have been reported previously for esterases (in- cluding subtilisin) in organic media, and we include our data here for comparison with other work. 16 As the concentration of alcohol is increased, the apparent K m for the ester also increases. Thus, one is limited in the range of substrate concentrations that can provide useful information. Also, as alcohol concentration increases, the physical properties of the solvent are al- tered, and we have seen deviation from expected behavior. Once again, the conclusion of this experimental approach is that the acyl-enzyme mechanism is ob- served by subtilisin in anhydrous tetrahydrofuran. The mechanism has also been tested by comparing the results of saturation kinetic studies in the presence and absence of added nucleophile. In Table 6, kcat/K m values, as determined from saturation kinetics, are reported for three substrates.

Esterases such as lipases and proteases are considered to catalyze transesterification in organic media via the acyl-enzyme mechanism. Zaks and Klibanov ]7 and Mitsuda and Nabeshima 18 have analyzed the mechanism of lipase-catalyzed transesterification reactions via the double-reciprocal parallel-lines method. Data are also available for the effect of nucleophile concentration on the rate of peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin solubilized in benzene. 19 To our knowledge, raw data for subtilisin- catalyzed transesterification reactions have not been published previously.

Conclusions The results of the steady-state kinetic studies reported above formally substantiate the presence of the acyl- enzyme mechanism for subtilisin-catalyzed transesterification reactions. Partitioning experiments in organic media indicate the formation of a common acyl-enzyme complex during subtilisin-catalyzed transesterification reactions in organic media. The fit of experimental data to kinetic models developed on a hypothesized acyl- enzyme mechanism may be considered as proof of the acyl-enzyme mechanism for transesterification in organic media. The occurrence of a partial reaction between subtilisin andp-nitrophenyl butyrate implies that transesterification follows the ping-pong mechanism. Interestingly, this partial reaction can be used to de- velop an active-site titration method for subtilisin in organic solvents.

Acknowledgements We gratefully acknowledge contracts and gifts from the University of Pittsburgh Materials Research Center,

1028 Enzyme Microb. Technol. , 1993, vol. 15, December

Subtilisin in organic so~vents: S. Chatterjee and A. J. Russell

Department of Defense (Army Research Office; DAAL03-90-M-0276), the Union Carbide Corporation, Eastman Kodak, PPG, the American Chemical Society (PRF Type G), and a National Science Foundation Presidential Young Investigator Award to A.J.R. (BCS 9057312), without which this research would not have been possible. S.C. was supported by the Department of Chemical and Petroleum Engineering at the Univer- sity of Pittsburgh.

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kinetic analysis of the mechanism for subtilisin in essentially anhydrous organic solvents

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