Jan 19, 1984 - saturated ammonium sulfate using chemically unmod- .... buffer and 1 ml of hydrazine sulfate (3.5 mM) in 0.1 M TEA buffer. (assay B). Aldehyde ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.
Vol. 259, No. 16,Issue of August 25, pp. 10222-10227.1984 Printed in U.S.A.
Catalytic Activity of Rabbit Skeletal Muscle Aldolasein the Crystalline State* (Received for publication, January 19, 1984)
J. SyguschS andD. Beaudry From the Departement de Biochimie,Faculte de Medecine, Uniuersite de Sherbrooke, Fleurimont, Quebec, Canuda J l H 5N4
Themonoclinic crystalline formofaldolasefrom lytic activity has been attributed tosteric hindranceproduced rabbit skeletal ‘muscle grown at 29 “C is catalytically by packing of the enzyme in the crystalline state (7-lo), to active in the direction of aldol cleavage. Activity was diffusion effects due to thesize of crystals used for the activity assayed forin a crystallization buffer containing46% study (6), or to inhibition of catalytic activity by components saturated ammonium sulfate using chemicallyunmod- or conditions of the crystallization buffer. Recovery of cataified single crystals cut to precise dimensions. Diffu- lytic activity in the crystalline state is possible by several sion effects on velocities from assays employing aldo- means. Microcrystalline enzyme preparations, for instance, lase crystals do not appear to be limiting when cut can be used to eliminate diffusion effects. Recrystallization of single crystals are crushed. Assaysof crushed crystals enzyme can be effected in buffers which conserve catalytic are linear with respect to both time and enzyme con- activity and/or which modify steric hindrance by inducing centration. Kinetic constants are reported for both sub-differential packing of the enzyme in the crystalline state. strates fructose1-phosphateandfructose 1,6-phosphate. Maximalvelocities and binding constants deter- Nevertheless, it is possible that because of the highly conmined differ by no morethan a factorof 2 between the densed state in which the enzyme packs, the crystallized crystalline and the soluble state of the enzyme. Anal- enzyme favors certain conformational state(s) that are not ysis of the kinetic constants for fructose 1-phosphate preferred in the solution environment. This can be of signifas substrate shows that binding of substrate does not icance especially in cases where enzymes in vivo are sequeschange in going to the crystalline state. Release of tered in distinct physical compartments at high concentraproduct is reduced roughly 2-fold in the crystalline tions or are immobilized on various molecular supports. As part of a studyto relate the molecular architecture being state. A similar conclusion can be reached in thecase determined by x-ray crystallography of aldolase from rabbit of fructose 1,g-phosphate as substrate provided the “on” stepsof substrate and product are only diffusion skeletal muscle to the solution structure, the study of the limited but independent of the physical state of the catalytic activity of aldolase in the crystalline state hasbeen enzyme. Itis not possible to distinguish between a more carried out. To assay conformational states of crystalline sluggishconformationalchangeduring catalysis or aldolase, we propose to compare some of the elementary rate simply tighter product binding in thecrystalline state constants describing the catalytic cycle which have been deas compared to the soluble enzyme state. rived from crystalline and solution kinetic data. MATERIALSANDMETHODS
Studies attempting to relate the molecular architecture of an enzyme to its function are confronted by a fundamental problem. The detailed molecular structure of an enzyme is obtained from an x-ray analysis of crystals of the enzyme. Enzyme function is analyzed from kinetic parameters using solutions of the enzyme. The problem is thus one of reconciling an essentially static view of the enzyme in ahighly ordered condensed state (crystalline) withthat of a dynamic interpretation of enzyme behavior in a dilute solution environment. The relation between structure and function is facilitated if it can be shown that the enzyme in the crystalline state can undergo many of the same conformational changes as in solution. One way to assay for conformational changes in the crystalline state is to compare catalytic activityof the enzyme in both crystalline and solution state. Work on a varietyof crystallized proteolytic enzymes (1-6) has indicated that each of the enzymes is catalytically active to varying degrees in the crystalline state. Diminished cata* This investigation was supported by Grant MA-8088 from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom requests for reprints should be addressed.
Rabbit muscle aldolase, bovine serum albumin, a-glycerophosphate dehydrogenase, triose phosphate isomerase, glutathione, and NADH were purchased from Boehringer Mannheim Ltd. Purity of enzymes was verified by high loading of the protein on sodium dodecyl sulfatepolyacrylamide gels and developing the protein by the silver-staining technique available from Bio-Rad Laboratories. Hydrazine sulfate, Fru-1-P’, Fru-Pp, and dihydroxyacetone-P were purchase from Sigma and utilized without further modification. Aldolase velocities were measured by following either aldehyde production (11) or NADH oxidation (12). NADH oxidation was monitored by adding 50 pl of 0.1 M TEA buffer (pH 7.4, 0.1 mM EDTA) containing aldolase (2 pg/ml or less) to a solution which has previously been made up to 950 p1 in a cuvette by addition of 100 pl of Fru-P2 (variable concentration), 100 p1 of NADH (15mM), 100 pl of TEA buffer, pH 7.4 (0.5 M), 2.5 pl of triose phosphate isomerase (10pg), 2.5 pl of a-glycerophosphate dehydrogenase (10 pg), and 645 p1 of water (assay A). Aldehyde production was measured by addition of 100 pl of 0.1 M TEA buffer (pH 7.4, 0.1 mM EDTA) containing aldolase (2 pg/ml or less) to a cuvette previously made up t o 1.5 ml containing 500 p1 of Fru-Pz (variable concentration) in 0.1 M TEA buffer and 1 ml of hydrazine sulfate (3.5 mM) in 0.1 M TEA buffer (assay B). Aldehyde production in crystals (-2 pg of aldolase) was assayed by making up all solutions with 45% saturated ammonium The abbreviations used are: Fru-1-P, fructose 1-phosphate; FruPz, fructose 1,6-bisphosphate; dihydroxyacetone-P, dihydroxyacetone phosphate; glyceraldehyde-3-P, glyceraldehyde 3-phosphate; TEA, triethanolamine HC1.
10222
Catalytic Activity of Monoclinic Aldolase
Crystals
10223
No differences could be discerned on the basis of intensities from x-ray precession photographs taken of the h01 zone before and after kinetic assays. No protein was solubilized during the course of the assay as judged by assay of the supernatant which had been previously dialyzed to remove substrate and product molecules (curue b, Fig. 1).To ascertain whether a larger surface area improved solubilization of the protein from the crystal during the course of the assay, the previous crystal was crushed to microscopic size and activity of the dialyzed supernatant was determined after assaying the crushedcrystal (curue c, Fig. 1) in the usual manner. No protein could be detected on the basis of the velocity assay (curue d, Fig. 1). To ensure that any reduction in specific activity of crystalline aldolase was not due to the possibility that aldolase was partially denatured or inactivated in the c r y s t a l h e form, the same crushed crystal utilized for the preceding assays was dialyzed and then dissolved in assay buffer B (assay buffer C but without 45% saturated ammonium sulfate).Specific activity of the dissolved protein (curve e, Fig. 1) was the same as that for the solubilized enzyme under identical assaying conditions. Since the protein crystallizes in thepresence of the assayingbuffer C, to determine the effect of ammonium sulfate,which is required for crystallization, on the catalytic activity, the previously dissolved protein was dialyzed against assay buffer C and the activity was measured (curve f, Fig. 1).The specific activity was found to be slightly less than that of the soluble enzyme in the absence of ammoniumsulfate. Aggregation of the soluble enzyme during assay conditions should not pose a problem because of high dilution of the enzyme in the reactionbuffer ( 5 2 pglml). Nonspecific loss of the soluble enzyme was examined by adding to all assay buffers 1%(w/v) bovine serum albumin. No noticeable difference could be detected in the RESULTS outcomes of the experiments between the presence and the Crystals of the monoclinic form of aldolase when incubated absence of bovine serum albumin in thereaction buffer. in assaying buffer C display demonstrable catalytic activity Contrary to most instances, microcrystalline preparations (curue a, Fig. 1).The single crystal utilized for kinetic assays of rabbit muscle aldolase are not always obtainable. Microis not visibly changed during thecourse of the kinetic assay. crystalline preparations are generally employed to eliminate problems associatedwith diffusional retardation of substrates and products in and out of crystals. Some microcrystalline preparations with smallest dimensions up topm10have been reported freeof diffusion effects(10). To eliminate diffusional effects on velocities in the activity studies, cut single crystals e of aldolase were crushed. The maximum dimensions of the crushed crystalswere no greater than15Fm with the majority f of fragments being of 2-pm average dimension.The K , values for Fru-Pz as a substrate are considerably smaller by several -4orders of magnitudethanthe K,,, values forthe various a c enzymes for which crystalline activity studieshave been carried out. To ascertain the importanceof diffusional effects on a the catalytic activity using Fru-Pz as substrate, the specific activity calculated from crushed crystalswas compared to the specific activity of crystals cut toprecise known dimensions (Table I). A similar comparison of specific activity was made 0 using Fru-1-P as substrate (Table I); the K , of Fru-1-P is comparable to theK , of substrates frompreviously reported studies. FromTable I, thecatalyticactivityexhibited by 0 5 10 aldolase crystals is nota function of surface area andbecomes diffusion controlled when minimum overall dimension of the TIME(min.) FIG. 1. Continuous recording of absorbances at 240 nm em- crystals exceeds 50 pm. If crystal surface area controlsspecific ploying the hydrazine assay (buffer C) to detect enzymatic activity, then a cut crystal of 200-pm equidimension would activity of crystalline and soluble aldolase. a, activity assay from haveapproximatelyone-fourththe specific activitycorrea 0.1 X 0.1 X 0.1 mm crystak b, activity of dialyzed supernatant of u; sponding to a cut crystal of 100 pm equidimension whereasin c, activity of crystal used in (I but crushed; d, activity assay of dialyzed fact no catalytic activity was measurable during the assaying supernatant of crushed crystal; e, activity of crushed crystal in c dissolved in assay buffer B; f , activity of soluble aldolase from e period for the 200-pm crystal. From Table I, specific activity dialyzed against crystallization buffer C. does not significantly vary when crystal minimum dimensions sulfate which was purchased from Bethesda Research Laboratories (assay C). No influence of ammonium sulfate was detected on the hydrazine reaction. In each case, the change in absorbance at 340 nm for assay A and 240 nm for assays B and C was recorded immediately by using a Cary model 20 double beam spectrophotometer. Blanks used contained the entire assay system with aldolase being omitted. Aldolase activity is expressed as micromoles of Fru-P2 cleaved/min/mg of enzyme at 25 "C. All reactions were carried out in siliconized glass- and plasticware. Enzyme concentration was determined by assuming an A m of 1.02 for a 1mg/ml aldolase solution (13). NADH substrate concentrations were determined by using a molar extinction coefficient of 6.22 X IO3 M" cm" at 340 nm (14). Aldehyde concentrations were measured by absorbance of the hydrazone formed at 240 nm by reaction of glyceraldehyde-3-P and hydrazine sulfate using a molar extinction coefficient found to be 2.73 X lo3 M" cm". Crystals of the monoclinic form of rabbit skeletal muscle aldolase were grown in small test tubes (500 pl) from 45% saturated ammonium sulfate solutions at 29 "C. Crystallization buffer consisted of aldolase (5 mg/ml final concentration), 0.1 M TEA buffer, pH 7.4, 1 mM GSH, 1 mM EDTA, and 40 mM NaCI. Large crystalline plates appeared within a few days. For activity studies, crystalline plates were cut to0.1 X 0.1 X 0.1 mmdimensions, crushed to microcrystalline size, and then dialyzed overnight against 0.1 M TEA buffer, pH 7.4, 1 mM EDTA. Activity studies on crystals were carried out by allowing crystals to sink to thebottom of the reaction cuvette. For product inhibition studies, the reaction mixture with substrate omitted was incubated with dihydroxyacetone-P prior to recording. Analysis of the velocity data in terms of maximal velocity and binding constants was carried out using a nonlinear regression procedure (NLIN) available from SAS Institute Inc. The kinetic parameters and maximal velocities and estimates of their standard deviations (shown in parentheses) were determined from the kinetic model employed for direct plots. Errors in the velocity measurements were found to be on the average 2% of the measured velocity for the concentration ranges employed.
1'
3
I
Catalytic Activity of Monoclinic Aldolase Crystals
10224
TABLEI Effect of crystal size on actiuity of m u c k aldolase Substrate and crystal size
Specific activity
Relative activity
Relative area
1.00 0.97
2 1 1.66
pm
Fru-Pz" Crushed" (€15) -2 50 X 50 X 50b -2 100 x 100 x 100 -2 4 200 x 100 x 100 8 200 x 200 x 100 200 x 200 x 200 16 Fru-1-P Crushed" ( 4 5 ) 0.061 -2 -2 50 X 50 X 50b -2 100 x 100 x 100 ~~
4.51 4.38 3.36 2.07 0.58
0.74 0.46 0.13
2.66
4
-d
1.00 0.061 0.046
1.00 0.76
2
1 A crystal of muscle aldolase having original dimension of 100 X ~
~
100 X 100 pm was crushed. The largest dimension of any crushed fragment being always less than 15 pm with the majority of fragments being of average dimension
