ARCHIVES OF BIOCHEMISTRY Vol. 187, No. 1, April

AND BIOPHYSICS 15. pp. 163-169, 1978

Conformational

Stabilization

MARGUERITE Department

of Biochemistry

of Enzymes

VOLINI’ and Biophysics,

AND

in Covalent

SHU-FANG

University

of Hawaii,

Catalysis’

WANG Honolulu,

Hawaii

96822

Received August 8, 1977; revised December 2, 1977 The enzymes aspartate aminotransferase, rhodanese, and chymotrypsin form covalent substituted-enzyme intermediates during the course of their catalysis. The present analyses show that, in these covalent intermediates, the enzyme proteins are stabilized against pHinduced structural transitions to inert forms that occur in the free enzyme species and other forms not covaiently substituted

The reaction pathways for the three enzymes, aspartate aminotransferase (EC 2.6.1.1), rhodanese (thiosulfate-cyanide sulfurtransferase, EC 2.8.1-l), and chymotrypsin (EC 3.4.4.5). are shown in Fig. 1. All of these enzymes function by double-displacement mechanisms in which covalent substituted-enzyme intermediates are formed (l-8). In each of the pathways as presented, the formation of the covalent intermediate occurs as the second reaction step. For the aminotransferase, which utilizes the coenzyme pyridoxal phosphate, the covalent substituted-enzyme intermediate is the pyridoxal enzyme, E-PLP3 (Fig. la). In the course of this reaction, the pyridoxamine enzyme, E. PMP, combines with the amino-acceptor substrate, a-ketoglutarate, to form an addition complex which decomposes, discharging the product, Z-glutamate, and forming the covalent intermediate (steps 1 and 2). The covalent intermediate subsequently reacts with the amino-donor substrate, I-aspartate, to form a second addition complex which decomposes, discharging the second product, oxalacetate, and regenerating the E. PMP intermediate (steps 3 and 4). By way of contrast, in the E. PMP intermediate the pyridoxamine 1 This work was supported by Research Grant BMS 75-23299 from the National Science Foundation. ’ To whom reprint requests should be addressed. ‘Abbreviations used: E-PLP, pyridozal enzyme; E PMP, pyridozamine enzyme;, E, enzyme; DIP, diisopropylphosphoryk CD, circular dichroism; ORD, optical rotary dispersion; MRW, mean residue weight.

moiety is bound to the apoenzyme in large part by salt linkages. In E-PLP the pyridoxal moiety is bound, in addition, by an aldimine linkage. In the rhodanese-catalyzed reaction the covalent enzyme-sulfur intermediate, E-S, is formed by discharge of the product, sulfite ion, from an addition complex between the free enzyme and the donor substrate, thiosulfate ion (Fig. lb, steps 1 and 2). The acceptor substrate, cyanide ion, combines with the E-S intermediate to form the second product, thiocyanate ion, thereby regenerating the free enzyme. No kinetically significant addition complex is observed with cyanide ion under common experimental conditions (14). With the amino- and sulfurtransferases, the covalent intermediates are isolated by omitting from the reaction mixtures the substrates responsible for their decomposition. Since, in the case of chymotrypsin, this substrate is a water molecule (Fig. lc), other means of producing stable covalent intermediates are commonly used (8-10). In this work the productive covalent intertrimethylacetyl-chymotrypsin, mediate, was selected for study because of its relatively low rate of deacylation (step 3). It is formed by reaction of the free enzyme with the substrate, p-nitrophenyltrimethylacetate (steps 1 and 2). Results with this intermediate were compared with those obtained with the nonproductive covalent intermediate, diisopropylphosphoryl-chymotrypsin. The latter is formed by reaction 163 0003-9861/78/1871-0163$02.00/0 Copyright 0 1978 by Academic Press, Inc. Au rights of reproduction in any form reserved.

164

VOLINI AND WANG

Icompl*x,l

Icomplax,)

I

I

Gl”LEPLPLLASP

Cl bl “+-yz--E’ ISW r-SCN-

0%

1

IE.SSO;i

ho.

w+ - I4 x--E’

pN?-TMA

3

-I-fE.pNP-TMAI IE&

E-rA 31

1. Reaction pathways for (a) aspartate aminotransferase, (b) rhodanese. and (c) chymo-

trypsin. The overall reactions are: (a) a-ketoglutarate + 1-aspsrtate~ (b) SSO3x- + CN-5

Gglutamate + oxalacetate;

SO?- + SCN-; and (c) p-nitrophenyltrimethylacetate + Hz03 triE diisopropylphosphoryl-enzyme

methylacetate + p-nitrophenol or diisopropyltluorophosphate + F-.

of the free enzyme with the irreversible inhibitor, diisopropyh’iuorophosphate (steps 1 and 2). StructuraI studies related to those reported here have been described previously with these chymotrypsin intermediates (8-10). In the present report, the conformational stabilities to changes in pH are examined for ah of these covalent enzyme intermediates. They are compared with those for the enzyme forms indicated in Fig. 1, which undergo structural transitions to inert conformers designated as E’. MATERIALS

AND METHODS

Enzyme preparations. Aspartate aminotransferase wss purchased from the Sigma Chemical Co. as a suspension containii 3 M ammonium sulfate, 50 nnu maleate, and 2.5 mu a-ketoglutarate. Ten milliliters of the suspension was centrifuged. The pellet was dissolved in 4 ml of 0.5 mM sodium acetate buffer. It was subsequently dialyzed against two l-liter changes of 0.05 rnr+rsodium acetate buffer, pH 5.8. For most of the spectral experiments, 0.05ml aliquots of this solution were diluted either with 0.3 ml of Tris-acetate buffer, pH 8.3, 30 mM in acetate, or with 0.3 ml of 10 mM sodium acetate, pH 4.0. The stock enzyme solution was assayed before and after spectra were recorded. Except for specific activity measurements, the assay mixture contained 1 ml of Tris-acetate buffer (pH 8.3,

ionic strength 0.4), 30 pmol of a-ketoglutsrate (neutralized), 60 amol of Caspartate (neutralized), in a tinal volume of 3.0 ml. For specific activity measurements, the reaction mixtures were the same as specified by Sizer and Jenkins in Method II (11). Rates were measured by following the appearance of oxalacetate absorbance at 280 nm. Protein concentrations were estimated by a biuret method (12). The specific activity at 37% was 2.9 U/min/mg, according to Method II reported by Sizer and Jenkins (11). Crystalline beef liver rhodanese was prepared by the method of Horowitz and De Toma (13) with the modifications described previously (14). Protein and activity measurements were the same as given previously (14). For spectral measurements, the stock protein solution was diluted with either Na-K phosphate buffer, pH 7.0, or Tris-sulfate buffer (ionic strength 0.1, pH 8.6). Crystalline a-chymotrypsin and diiiopropylphosphoryl (DIP)-chymotrypsin were obtained from the Worthington Corp. Trimethylacetyl chymotrypsin wss prepared from a-chymotrypsin and recrystallixed p-nitrotrimethylacetate. Stock solutions in 0.1 M KC1 at pH 3.55 were diluted with Tris-acetate buffers, 0.1 ionic strength, for the spectral measurements. Protein concentrations and enzyme activity were measured as described previously (10). Spectral determinations. Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra were recorded on a Cary Model 60 spectropolarimeter with CD attachment. Absorption spectra were recorded on a Cary Model 15 spectrophotometer. For the amino-

CONFORMATIONAL

STABILIZATION

transferase, protein concentrations varied from 1.0 to 2.0 mg/ml. For rhodanese and chymotrypsin, the protein concentration was 0.9 mg/ml. The samples were examined in cells of O.l- or 0.5~mm path length at a sensitivity of 0.1 or 0.04” full scale. The ellipticity values (@MRW for rhodanese and chymotrypsin were calculated using a mean residue weight of 115. The reduced molar rotation values (m’)uaw for the aminotransferase were corrected for solvent refractive index. They are based on a mean residue weight of 114 (15). RESULTS

Aspartate aminotransferase. The sequence shown in Fig. la was considered as two half-reactions at equilibrium. The relative concentrations of the pyridoxal and pyridoxamine intermediates, E-PLP and E. PMP, were varied by adding different concentrations of cy-ketoglutarate and dlglutamate to solutions of the enzyme initially in the E-PLP form. In the diagram, (complexr) is meant to represent both the (E-PLP . GLU) complex and its isomerized form (E . PMP .~YKG). As pointed out by Velick and Vavra (l), at low substrate concentrations there is no appreciable accumulation of the complexes, and the ratio of the intermediates is given by the expression. K=KIKz=

(Glu) (E - PLP) ((w- KG) (E.PMP) ’

where KI and KZ are the equilibrium constants for steps 1 and 2 in Fig. la. At pH 8.3 the relative concentrations of the two intermediates were estimated, using as a guide the values of the constants determined kinetically by Velick and Vavra (1) and Henson and Cleland (2). In Fig. 2 (right), the ORD spectrum of the enzyme treated with a-ketoglutarate at pH 8.3 is compared with that of the enzyme treated with a-ketoglutarate at pH 4.0. As shown, no significant difference was observed, indicating that the conformation of the covalent pyridoxal intermediate is unchanged by the alteration in pH. The same conformation was observed with untreated enzyme solutions, in accordance with the fact that the enzyme was initially in the pyridoxal form. The ORD spectrum of the enzyme treated with &glutamate at pH 8.3 (Fig. 2,

165

OF ENZYMES

left) was essentially the same as that observed for the enzyme treated with cr-ketoglutarate. However, upon treatment with d&glutamate in concentrations of 1.4-4.2 mu at pH 4.0, decreases in the reduced molar rotation to 3900 deg-cm2 dmol-’ were observed at 199 nm (Fig. 2, left). This represents a 14% change in the rotation observed at pH 8.3. Since the experimental error was

Conformational stabilization of enzymes in covalent catalysis.

ARCHIVES OF BIOCHEMISTRY Vol. 187, No. 1, April AND BIOPHYSICS 15. pp. 163-169, 1978 Conformational Stabilization MARGUERITE Department of Bioche...
514KB Sizes 0 Downloads 0 Views