Bkconlugete Chem. 1881, 2, 211-216
211
Introduction of Unnatural Amino Acids into Chalcone Isomerase Rodney A. Bednar,’ Catherine McCaffrey, and Kaiyu Shan Departmenta of Pharmacological Sciences and Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-8651. Received March 19,1991;Revised Manuscript Received May 7,1991 The active site cysteine residue of chalcone isomerase was rapidly and selectively modified under denaturing conditions with a variety of electrophilic reagents. These denaturated and modified enzyme were renatured to produce enzyme derivatives containing a series of unnatural amino acids in the active site. Addition of methyl, ethyl, butyl, heptyl, and benzyl groups to the cyteine sulfur does not abolish catalytic activity, although the activity decreases as the steric bulk of the amino acid side-chain increases. Modification of the cyteine to introduce a charged homoglutamate or a neutral homoglutamine analogue results in retention of 22% of the catalytic activity. Addition of a methylthio group (SMe) to the cysteine residue of native chalcone isomerase preserves 85% of the catalytic activity measured with 2’,4’,4-trihydroxychalcone,2‘,4’,6’,4-tetrahydroxychalcone, or 2‘-hydroxy-4-methoxychalconeas substrates. The competitive inhibition constant for 4’,4-dihydroxychalcone,the substrate inhibition constant for 2‘,4‘,44rihydroxychalcone,and other steady-state kinetic parameters for the methanethiolated enzyme are very similar to those of the native enzyme. The strong binding of 4’,4-dihydroxychalcone to the methanethiolated enzyme shows that there is no steric repulsion between this modified amino acid residue and the substrate analogue. This structure-activity study clearly demonstrates that the active site cysteine residue does not function as an acid-base or nucleophilic group in producing the catalysis or substrate inhibition observed with chalcone isomerase. The method presented in this paper allows for the rapid introduction of a series of unnatural amino acids into the active site as a means of probing the structure-function relationship.
INTRODUCTION
Hoflo
Chart I
A basic tenet of biochemistry is that structure determines function. To understand this structure-function relationship, we must have tools that allow us to produce enzyme molecules with defined altered structures. Using the enzyme chalcone isomerase (EC 5.5.1.6), we illustrate a method to rapidly introduce a series of unnatural amino acid residues in place of a cysteine residue and use this method to probe the role of hydrophobic and charge interaction in binding and catalysis. Chalcone isomerase catalyzes the cyclization of 2’,4’,4trihydroxychalcone (I) to 4’,7-dihydroxyflavanone (11) (Dixon et al., 1982, 1988; Ebel & Hahlbrock, 1982) (see Chart I). Although several mechanisms have been suggested for how the enzyme catalyzes this simple additionelimination reaction (Hahlbrock et al., 1970; Boland & Wong, 1979), little is known about the role of active-site residues. Chalcone isomerase loses activity upon treatment with iodoacetamide (Boland & Wong, 1975),N-ethylmaleimide (Bednar, 1990),and mercurials (Moustafa & Wong, 1967; Dixon et al., 1982; Bednar et al., 1989). The single cysteine residue in the chalcone isomerase from soybeans (Glycine m a ) is located a t or near the active site, but ita function has not yet been unequivocally defined (Bednar et al., 1989). In order to probe the role of the active site cysteine residue of chalcone isomerase we have produced enzyme derivatives in which the cysteine residue has been converted to a series of unnatural amino acids. The results presented in this paper clearly demonstrate that the cysteine residue in chalcone isomerase does not function as an acid-base or nucleophilic group during catalysis nor does it play any role in the substrate inhibition observed for this enzyme. Further, the series of unnatural amino
* To whom inquires and reprint requests should be addressed. lQ43-18Q2l91/29Q2-Q211$02.50/0
----c c
\6’
6‘
0
acids introduced into chalcone isomerase allow us to probe the effects of adding hydrophobic and charged groups into an active site. EXPERIMENTAL PROCEDURES Materials. MMTS,’ p(hydroxymercuri)benzoate,BSA, 4-(iodoacetamido)salicylicacid, iodoacetamide, iodoacetate, and naringenin were purchased from Sigma. Methyl iodide, bromoethane, iodoheptane, and benzyl bromide were obtained from Aldrich. Bromobutane was obtained from Eastman. 2’-Hydroxy-4-methoxychalcone was obtained from the Alfred Bader Library of Rare Chemicals (Aldrich) and the purity was determined to be greater than 98% by HPLC analysis. 2’,4’,6’,4-Tetrahydroxychalconewas prepared from naringenin by using the method of Moustafa & Wong (1967). The 4’,4-DHC and 2’,4’,4-trihydroxychalcone were synthesized and purified as described previously (Bednar & Hadcock, 1988; Geissman & Clinton, 1946). Stock solutions of flavonoidswere prepared in 95% Abbrevations used BSA, bovine serum albumin; 4’,4-DHC,
4’,4-dihydroxychalcone; DTT,dithiothreitol; EDTA, ethylenediaminetetraaceticacid; NEM, N-ethylmaleimide;pMB, p ( h y droxymercuri)benzoat;MMTS,S-methyl methanethioeulfonata;
CHES, 2-(cyclohexylamino)ethanesulfonicacid; HEPES, N42hydroxyethyl)piperazine-N‘-2-ethanesulfonicacid; MES, 2-Nmorpholinoethanesulfonic acid; MOPSO, 3-N-morpholino-2hydroxypropanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane. Q 1991 Amerlcan Chemlcal Soclety
Bednar et el.
212 Bkconlugate Chem., Vol. 2, No. 4, 1991
EtOH. Other materials and methods have been described previously (Bednar & Hadcock, 1988;Bednar et al., 1989). Chalcone isomerase was isolated from Williams-82 cultivar of soybeans (Glycine m a ) and purified to homogeneity (Bednar & Hadcock, 1988). The standard assay for chalcone isomerase activity was performed by monitoring the loss of the chalcone absorbance at 390 nm at 25 “C using 40 pM 2’,4’,4-trihydroxychalconein 50 mM Trischloride buffer (pH 7.6) containing 1%EtOH (Bednar & Hadcock, 1988). One international unit of enzyme activity is defined as the amount of enzyme that catalyzes the loss of 1pmol of 2’,4’,4-trihydroxychalconein 1min at 25 “C in 50 mM Tris-chloride, pH 7.6. The concentration of active enzyme was calculated from the observed activity (IU/mL) using a molecular weight of 24 OOO and a specific activity of 340 IU/mg (Bednar & Hadcock, 1988). Treatment of Chalcone Isomerase with Alkylating Agents. Stock solutions of iodoacetamide (1M), iodoacetate (1 M Na salt), 4-(iodoacetamide)salicylic acid (0.1 M titrated with KOH), sodium tetrathionate (0.1 M), NEM (0.1 M),and MMTS (0.1 M) were freshly prepared in Milli-Q water. The alkyl halides methyl iodide, bromoethane, bromobutane, iodoheptane, and benzyl bromide were used neat. Chalcone isomerase (0.18 pM) was incubated at pH 7.5 with a 100-fold dilution of the alkylating agent in 5 M urea, 50 mM HEPES containing 0.1 mg/mL BSA and 1 mM EDTA. After a 5-10-min incubation a t 25 OC, the enzyme was renatured by a 20fold dilution into 50 mM HEPES a t pH 7.5 containing 0.1 mg/mL BSA and 1 mM EDTA. The activity of the modified enzyme was measured by the standard assay procedure (E)and also with assay solution supplemented with 1 mM pMB ( E + p ~ A ~ )control . incubation lacking the alkylating reagent was run in parallel and its activity determined by the standard assay procedure (Ed.Native enzyme is instantaneously inactivated in the presence of 1mM pMB, while the activity of cysteine-modifiedenzyme is insensitive to the presence of pMB. The activity observed in the presence of pMB depends on the catalytic activity of the modified enzyme (EM)and the fraction of the enzyme modified ( f ~ (eq ) 1). The activity observed E+pMB
= EbdM
(1)
in the absence of pMB has an additional contribution from any unmodified enzyme (Ec) remaining (eq 2). E
EdM + E,
(2)
(1-fM)
Rearrangement of eqs 1and 2 allows us to calculate the ) the relative fraction of the enzyme modified ( f ~ and catalytic activity of completely modified enzyme ( E M / Ec)according to eqs 3 and 4, respectively. fM
= E+PMB/~C -
+
(3)
= (E+pMB/EC)/fM
(4)
Kinetics of Modification of Chalcone Isomerase with MMTS. Chalcone isomerase (1.1pM) was incubated with MMTS (1mM) at 25 “C and pH 6.8 in 50 mM MCPSO buffer I = 0.1 M (KC1). Aliquots of enzyme were periodically removed and assayed as described above. For an irreversible reaction with a constant concentration of a modifying reagent, the fraction of the enzyme modified will follow a single exponential (eq 5). Substitution of eq fM
= 1- e ( - L d M m l r )
(5)
5 into eqs 1 and 2 yields a description for the time-de-
0.4
0.2--
\
order rate constant for modification ( k m d ) and the relative activity of completely modified enzyme (EM/Ec)were determined by a nonlinear least-square fit of the timedependent activity data according to eqs 6 and 7. Protection against Modification by 4’,4-DHC. Chalcone isomerase (0.27 rM) was incubated with MMTS (1 mM) containing 4’,4-DHC (0-210 pM) in 50 mM MOPS0 (50% base, I = 0.1 M KC1, pH 6.8). Aliquota were removed over time and assayed for enzymatic activity in the presenceand absence of pMB. Control incubations lacking 4’,4-DHC were run in parallel. The observed rate constants for modification, kmd, were obtained according to eqs 6 and 7. The binding constant for 4’,4-DHC a t the site from which it protects, Kp, and the rate constant for modification in the absence of protectant, ( k m d ) - , were determined by fitting a plot of log k m d as a function of [4’,4-DHC] according to eq 8. log kmd = log l ( k m d ) m a / ( l +
[4’,4-DHCl/Kp)J (8)
RESULTS
Effect of Urea on Enzyme Structure and Activity. Incubation of chalcone isomerase with greater than 5 M urea results in loss of the catalytic activity (Figure 1). However, essentially all the catalytic activity is rapidly regained by a 100-fold dilution of the mixture into assay solution (Figure 1). Incubation of chalcone isomerasewith iodoacetamide (0.8 M) results in a slow time-dependent loss in enzymatic activity. The loss of activity is biphasic (12 = 0.1 M-l min-l for the fast phase after correction for the slow phase) with little change in the time-dependence for inactivation between pH 6.6 and 9.2 (data not shown). When the incubation with iodoacetamide is performed a t pH 7.5 in 5 M urea, the cysteine residue is rapidly modified (kmd > 35 M-’ m i d ) to produce an analogue of homoglutamine with only partial loss in enzymatic activity (Table I). The denaturation of the enzyme in urea increases the rate constant for modification of the enzy-
Manlpulating ActiveSite Properties
Bbconju@?te Chem., Vol. 2, No. 4, 1901 213
Table I. Activity of Chalcone Isomerares containing an Altered Active Site Cysteine altered enzyme ES-H ES-SCHn ESCHs ESCH&Ha ESCHzC(0)NHz ESCHzC(0)OESCHpCHpCH2CHs ES-CHZCH~CH&HZCHCH~CH~ ES-CHaCar,
0 8
relative activity (EhalEc) 1.00 0.86
0.66 0.31 0.22 0.22 0.19 0.12 0.w co.01
U
E! 0
:E" 10
!:!ncnn
20 30 Time (min)
40
d500.0
ESH + Na2S408
