THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 29, Issue of October 15, pp. 17493-17498,lSSO 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Construction and Properties of Active Chimeric Enzymes between Human Aldolases A and B ANALYSIS OF MOLECULAR REGIONS WHICH DETERMINE ISOZYME-SPECIFIC FUNCTIONS*

(Received for publication, February 21, 1990)

Yoshihiko Kitajima, Yozo Takasaki, Isamu Takahashi, and Katsuji Hori$ From the Department of Biochemistry, Saga Medical School, Nabeshima, Saga 849, Japan

To study the structure/function relationships of hu- man aldolase isozymes, particularly isozyme-specific functions, we constructed Escherichia coli expression plasmids for six BA chimeric enzymes (BA34, BA108, BA137, BA212, BA306, and BA306*), each composed of the N-terminal side of isozyme B and the C-terminal side of isozyme A, and one BAB chimeric enzyme which contains a fragment of isozyme A (residues 213-306) inserted in between the N-terminal and the C-terminal fragments of isozyme B. They were transfected into E. coli, and the generated enzymes were characterized.

This study reveals the following. (i) For isozyme A, the C-terminal Tyr-363 and the N-terminal region bearing isozyme group-specific sequences l-3 and Lys-107 (the C-6 phosphate-binding site) are respon- sible for the higher catalytic activity toward fructose 1,6-bisphosphate, which is 7 times higher than that of aldolase B. Conversely, an internal region spanning positions 108-212 is required for the lower activity toward fructose l-phosphate. (ii) For isozyme B, an internal sequence spanning positions 108-212 which includes some isozyme B-specific residues and a pos- tulated C-l phosphate-binding site (Lys-146 or Arg- 148) is responsible for a higher catalytic activity to- ward fructose l-phosphate, which is 8-10 times that of isozyme A. The more upstream sequence containing positions l-107 is responsible for the lower catalytic activity toward fructose 1,6-bisphosphate. (iii) At least residues 212-306, composing a long stretch near the active-site Lys-229 and highly conserved among iso- zymes A, B, and C, may be required for the basal framework of the aldolase molecule to exhibit the ac- tivity common to the three isozymic forms.

There are three isozymic forms of vertebrate aldolase (EC 4.1.2.13), A (muscle type), B (liver type), and C (brain type) (1, 2). These isozymes show different specificities toward the substrates fructose 1,6-bisphosphate (Fru-1,6-P*)’ and fruc- tose l-phosphate (Fru-1-P); and therefore, the activity ratios of Fru-1,6-P* to Fru-1-P are approximately 50 for isozyme A, 1 for isozyme B, and 10 for isozyme C (2). Thus, the charac- teristics exhibited by these isozymes appear to be suited for

* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. 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 correspondence should be addressed. ’ The abbreviations used are: Fru-1,6-Pz, fructose l&bisphosphate;

Fru-l-P, fructose l-phosphate; CCS, conserved common sequence; IGS, isozyme group-specific sequence.

sugar phosphate metabolism in the respective tissues and would mainly be endowed by the isozyme-specific sequences on the molecule acquired through molecular evolution of the three isozyme genes after divergence. Accordingly, it is of interest to explore in this capacity which amino acid se- quence(s) on the molecule determines the respective isozyme- specific functions. Recently, the amino acid sequences of at least eight different vertebrate aldolase molecules were de- duced from nucleotide sequences of genomic and/or cDNA clones (3-IO), enabling us to identify the similarities and dissimilarities of amino acid sequences among the three iso- zymic forms. In addition, x-ray crystallographic analyses of rabbit and human aldolases A at 2.7- and 3.0-A resolutions, respectively, provided further information on the structure of the aldolase molecule (11, 12).

To date, several different approaches, e.g. chemical (13,14) and enzymatic (15, 16) modifications, site-directed mutagen- esis (17, 18), artificial hybrid formation (19), as well as anal- yses of altered enzymes from hereditary disease patients (20- 24), have been employed to determine the structure/function relationships of aldolase isozymes. In this study, we con- structed seven chimeric enzymes that shared partial amino acid sequences derived from their parent enzymes, isozymes A and B, and characterized these artificially constructed enzymes to determine which regions on the molecule are required for determining isozyme-specific functions. A similar approach has been described for phosphoglycerate kinases from humans and yeast (25). Here, we describe the construc- tion and properties of the chimeric enzymes. In addition, the role of isozyme group-specific sequences in determining the characteristics of these isozymes is discussed.

MATERIALS AND METHODS Bacterial Strains and Plasmids-Escherichia coli K12 strain JM83

from laboratory stock was used for cloning and expression experi- ments. pINI (26), and E. coli expression vector that contains a lipoprotein promoter (lpp), lac UV5 promoter operator (luc), and lactose repressor (loci), was provided by Dr. M. Kotani (Toyo Jozo Co., Ltd.). pHAAL116-3 (a cDNA clone of human aldolase A) and pHABL120-3 (a cDNA clone of human aldolase B), both of which were isolated in this laboratory as previously described (27), were used to construct the expression plasmids.

Reagents-Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and other enzymes were purchased from Takara Biochemicals, Nippon Gene, and Toyobo Co., Ltd. [a-“‘PI and the DNA sequencing kit were obtained from ICN Radiochemicals and Amersham Corp., respectively.

Purification of Human Aldolases Expressed in E. coli-E. coli JM83 carrying the plasmids was grown overnight in brain heart infusion (Difco) containing 50 pg/ml of ampicillin, and the cells were harvested as previously described (27). Normal isozymes A and B and chimeric enzymes expressed in E. coli were purified as previously described (17) by a modification of the method of Penhoet et al. (28) for vertebrate aldolase. All purification procedures were carried out at

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17494 Chimeric Human Aldolase Isozymes

4 “C in the presence of 1 mM phenylmethylsulfonyl fluoride unless otherwise indicated.

A.ssav of Enzyme Actiuity-Aldolase activity was assayed either by an actibity staining method (29) or by a spectrophotometric method (30). In this staining method, 10 &I of each enzyme preparation was applied to a cellulose acetate strip, subjected to electrophoresis for 40 min at 250 V at 4 “C, and stained as previously described (29); and 10 mM EDTA was added to inhibit endogenous bacterial class II aldolase activity unless otherwise indicated. The spectrophotometric assay was performed at 30 ‘C according to Rajkumar et al. (30) in the presence of either Fru-1,6-P? or Fru-1-P. One unit of aldolase activity is defined as that amount of enzyme which catalyzes the aldol cleavage of 1 rmol of Fru-1,6-P? or Fru-l-P/min at 30 “C, and the specific activity is defined as units of enzyme activity/milligram of protein.

Determination of Physicochemical Properties-The molecular sizes of aldolase were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (31). Those of the native tetrameric forms of aldolase were determined as previously described (32). CD spectra were obtained with a JASCO 5600 spectrophotometer.

RESULTS AND DISCUSSION

Comparison of Primary Structures of Vertebrate Aldolases

Comparison of the entire amino acid sequences of eight different aldolases (3-10) revealed that the aldolase molecule is composed of several short and long stretches of sequences: (i) those highly conserved through the three isozymic groups (conserved common sequence (CCS)), (ii) those conserved within a single isozymic group (isozyme group-specific se- quence (IGS), and (iii) sequences showing diversity (Fig. 1). There are seven CCSs among vertebrate enzymes (Fig. 1, boxes 2-7). Lys-229, an active-site residue that is implicated in the Schiff base formation with the C-2 carbonyl group of Fru-1,6-P? or Fru-1-P (2), resides on the longest CCS-5 (res- idues 165-239 corresponding to region D-e-E-f (see Fig. 1) of rabbit aldolase A) (11). Lys-107, the C-6 phosphate-binding site (33, 34), and Lys-146 or Arg-148, a postulated C-l phos- phate-binding residue (35,36), are on CCS-4. On the contrary, the sequences of residues 36-48, 57-72, and 87-102 are iso- zyme group-specific (CCSs-(1-3); Fig. 1, circles 1-3). Simi- larly, the C-terminal sequence of positions 311-363 is also isozyme group-specific (CCS-4; Fig. 1, circle 4). Based on x- ray crystallographic data, IGSs-(1-3) appear to be situated on the flanking cr-helices of the P-barrel that are in array on the periphery of the compact molecule of aldolase A (11, 12).

Construction of Chimeric Enzymes between Human Aldolases A and B

To test which part of the molecules determines isozyme group-specific functions, we constructed. E. coli expression plasmids for seven chimeric enzymes between isozymes A and B (Fig. 2). These constructions were transfected into E. coli as described under “Materials and Methods,” and the gener- ated enzymes were purified as previously described (17). BA34, BA108, BA137, BA212, BA306, and BA306*, a group of chimeric enzymes, shared various lengths of a fragment of isozyme B at the N-terminal side and of a fragment of isozyme A at the C-terminal side within the total number of 363 amino acids joined together at the position indicated in each enzyme name (Fig. 2A). In BAB, another chimeric enzyme group, a fragment of isozyme A spanning positions 212-306 was in- serted in between the N-terminal (residues l-211) and the C- terminal (residues 307-363) fragments of isozyme B (Fig. 2B).

Characteristics of Chimeric Enzymes

Enzymatic Properties

All chimeric enzymes examined were active and showed electrophoretic mobilities identical to those of parent iso-

FIG. 1. Comparison of amino acid sequences of vertebrate aldolase isozymes. Amino acids identical to rabbit aldolase A se- quences are marked with dashes. Regions residing in commonly conserved amino acid sequences are boxed and darkly shaded (boxes l-7). Similarly, isozyme group-specific residues are asterisked, boxed, and lightly shaded (circles Z-4). The active-site Lys-229 is indicated (#) in the sequence frame. The closed circles in the amino acid sequences of rat and human aldolases C indicate the positions of a single amino acid deletion for maximum matching. Regions in a-helix ((Y(Y(Y(Y-ati, I, AI-H2) and P-sheet (~~~~-@& a-h) structures of rabbit aldolase A (11) are diagramed over the sequence. Literature sources are as follows: aldolase A from rabbit (5), rat (3), and human (4); aldolase B from rat (6), human (7), and chicken (8); and aldolase C from rat (9) and human (10). In this assignment, the N-terminal Met residue was omitted from the sequences.

zymes A and B (Fig. 3). The molecular sizes of all chimeric enzymes coincided with the values expected from those of isozymes A and B generated in E. coli (data not shown). The enzymes have successively lost the characteristics of isozyme A and consequently gained properties of isozyme B. General properties of chimeric enzymes are summarized in Table I.

Chimeric Enzyme BA34-BA34 showed a similarity to iso- zyme A with respect to its K, value for Fru-1,6-P*. The k,,, for the Fru-1,6-P2 cleaving reaction was -80% that of isozyme A, whereas that for Fru-1-P slightly increased, resulting in a decrease in the activity ratio of Fru-1,6-P* to Fru-1-P to 31. The K, for Fru-1,6-PZ was reduced to half that of isozyme A, whereas the K,,, for Fru-1-P was reduced to <X3. These results show that chimeric construction BA34 exhibits, in part, the original properties of isozyme B, but is basically very similar to isozyme A. This may be attributed to the functional simi- larity of the N-terminal regions spanning positions l-34 of isozymes A and B (Fig. 1, region la). In BA34, the N-terminal fragment of isozyme A offered both the C-6 phosphate-bind-

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Chimeric Human Aldolase Isozymes

FIG. 2. Construction of E. coli expression plasmids harbor- ing cDNA encoding human aldolases A and B and chimeric proteins between isozymes A and B. Upper, restriction maps of human aldolase A and B cDNAs. A, construction diagrams of the expression plasmids for the six BA chimeric enzymes; B, construction diagram of the expression plasmid for the BAB chimeric enzyme. The coding regions of isozyme A and B expression plasmids and the corresponding amino acid sequences are represented by stipled and closed boxes, respectively. The noncoding regions of the expression plasmids are represented by open boxes. The numbers over the dia- gram of proteins represent the amino acid positions of the BA boundary. Arrowheads in expression plasmids represent E. coli lipo- protein (Ipp) and lactose (lac) promoters derived from the pINI vector as shown in a previous paper (17, 18). Thin lines in expression plasmids represent the sequence of the expression vector. AC, AccI; Bg, BglI; E, EcoRI; H, HindIII; Hf Hi&I; Es, RsaI. pHAA47, and E. coli expression plasmid for isozyme A, and pHAB141, an expression plasmid for isozyme B, were constructed as described in a previous paper (27). pHAA47-Y363S, an expression plasmid for an altered human aldolase A with substitution of Ser for Tyr at the C terminus (position 363), was constructed as previously described (17). Chimeric aldolase cDNAs were constructed by connecting cDNA fragments for isozymes A and B which were produced by digesting each of the inserts, pHAA47 and pHAB141, with restriction endonucleases at various sites common to both cDNAs. First, pHA-BA34, which was designed to encode a chimeric protein bearing the N-terminal 34 residues of isozyme B and the C-terminal 329 residues of isozyme A, was constructed as follows. A 103-base pair-long EcoRI-HinfI frag- ment of an isozyme B cDNA insert from pHABI41 which covered the sequence encoding the N-terminal 34 residues was ligated to a 1072-base pair-long Hi&I-Hind111 fragment of an isozyme A cDNA insert from pHAA47 which covered the sequence encoding the C- terminal 329 residues. The 1175-base pair-long EcoRI-Hind111 frag- ment thus generated was then inserted into an E. coli pUC13 cloning vector. The positive colony for this chimeric enzyme was screened directly by dideoxy DNA sequencing (37) and cloned again into an E. coli expression vector to construct expression plasmid pHA-BA34. Three other plasmids, pHA-BA137, pHA-BA212, and pHA-BA306, were constructed by procedures similar to those used for the construc- tion of pHA-BA34; each cDNA insert for isozymes A and B was cleaved by a restriction endonuclease as shown in this figure (RsaI for pHA-BA137, AccI for pHA-BA212, and BglI for pHA-BA306) to generate cDNA fragments which encode for a different number of residues of isozymes A and B. For construction of the fifth expression plasmid, pHA-BA108, an AccI site on isozyme B cDNA corresponding to that marked with an open star in isozyme A cDNA was created by changing a triplet TTA for Leu-108 to GTA for Val-108 by site-

BA306*

BA306 @

BA212 t

17495

+

4

BA108

BA34

A

FIG. 3. Zymograms of normal and chimeric human aldol- ases expressed in E. coli. Activity staining was carried out on an agar plate without EDTA according to Susor et al. (29). Activity spots in front of those for chimeric constructs and authentic human aldol- ases moving toward the anode are of E. coli class II aldolase. The activity spot of BA137 was only barely seen unless the concentrated preparation was subjected to electrophoresis. 0, electrophoresis ori- gin; + and -, anodic and cathodic sides, respectively.

ing site Lys-107 and the C-l phosphate-binding site Lys-146 (or Arg-148); and thus, the /?-barrel structure of this chimeric enzyme seems to be essentially very similar to that of isozyme A.

Chimeric Enzyme BAIO8-The tendency toward the B-type isozyme in nature was clearly enhanced in BA108, BA212, and BA306, which shared much longer N-terminal sequences of isozyme B. With chimeric enzyme BA108, the k,., for Fru- 1,6-P* was markedly reduced to less than one-fourth that of normal isozyme A, whereas the k,., for Fru-I-P still remained at a level only 2-fold higher than that of isozyme A, resulting in a decrease in the activity ratio of Fru-1,6-P* to Fru-1-P to 6.5. The K, for Fru-1,6-P, of this chimeric enzyme also decreased to an intermediate value between isozymes A and B. These results strongly suggest that, as Lys-107 is conserved among isozymes A, B, and C (Fig. l), a sequence(s) or resi- due(s) within positions 34-108 of isozyme A (probably IGS- (l-3), one, two, or all of them) may possibly play a modulating role in keeping the catalytic activity toward Fru-1,6-PP at a higher level.

Chimeric Enzyme BA212 and BA306-As compared to BA34 and BA108, chimeric enzymes BA212 and BA306 more closely resembled isozyme B with respect to the Fru-1,6-PP and Fru-1-P and the activity ratio of Fru-1,6-PZ to Fru-1-P. In addition, the K,,, values for Fru-1-P of these two chimeric enzymes dramatically increased to values almost comparable to that of isozyme B, whereas those for Fru-1,6-Pp inversely decreased to a level only 1.7-2.4-fold higher than that of

directed mutagenesis. The fragments derived from aldolase A and B cDNAs were ligated to generate a full-size cDNA encoding BA chi- merit protein having 363 residues fused at position 108, 137, 212, or 306. The sixth plasmid, pHA-BA306*, was constructed by ligating a EcoRI-BglI fragment of pHAB141 to a BglI-Hind111 fragment of pHAA47-Y363S (17). The seventh plasmid, pHA-BAB, was con- structed from an EcoRI-BglI fragment of pHA-BA212 and a BglI- Hind111 fragment of pHAB141. These constructions were confirmed to carry the entire nucleotide sequences for the respective aldolase cDNAs by the dideoxynucleotide chain termination method (37). The respective numbers of these chimeric aldolases represent amino acid positions of the chimeric boundary. Chimeric aldolases encoded by pHA-BA34, pHA-BA109, pHA-BA137, pHA-BA212, pHA-BA306, pHA-BA306*, and pHA-BAB are referred to as BA34, BAIOB, BA137, BA212, BA306, and BA306*, respectively.

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Chimeric Human Aldolase Isozymes

TABLE I Ch aracteristics of normal and chimeric human aldolases

K,,, values for Fru-1,6-P* (FBP) and Fru-1-P (FlP) of these en- zymes were determined by measuring activities in the presence of various amounts of substrates under standard assay conditions (30) as described in the legend to Fig. 4 and determined by a Lineweaver- Burk plot.

Aldolase

A BA34 BA108 BA137 BA212 BA306 BA306* BAB B

k cat

FBP FlP

min-’ 1856 32 1520 48

416 64 32 16

432 208 608 400 256 240 208 144 256 260

FBP/FlP

58 31

6.5 2.0 2.1 1.5 1.1 1.5 1.0

K”,

FBP FlP

M

8.0 x lo+ 7.1 x lo-” 4.0 x lo+ 2.0 x lo-” 2.3 x lo-” 8.3 x lo+ 6.7 x 1O-6 6.3 x lo+ 5.3 x 1o-6 5.0 x 1o-3 7.4 x 1o-6 2.6 x lo-” 8.0 x 1O-6 1.7 x 1o-3 4.4 x lo+ 2.5 x 1O-3 6.0 x 1O-6 2.5 x 1O-3

isozyme B. These results strongly suggest that a region(s) in residues 108212 of isozyme B is responsible for the higher catalytic activity toward Fru-1-P and the lower K,,, values for Fru-1,6-P* which are fairly comparable to those of isozyme B and thus the low Fru-1,6-PJFru-1-P activity ratio. As shown in Fig. 1, since a postulated C-l phosphate-binding site (Lys- 146 or Arg-148) and the region(s) surrounding the residues are highly conserved in isozymes A, B, and C, any one or some of the residues near the C-l phosphate-binding site, e.g. Leu-108, Ala-113, Ile-124, Gly-145, Ala-149, Gln-164, Leu- 182, and Val-190, or the short stretches including these hy- drophobic residues in isozyme B could be responsible for maintaining the higher catalytic activity toward Fru-1-P that characterizes isozyme B. The importance of the conservation of Ala-149 in isozyme B was proven by the catalytic defect of human aldolase B from several patients with hereditary fruc- tose intolerance with the Ala-149 + Pro-149 substitution (22).

Chimeric Enzyme B306*-BA306* is a derivative of BA306 which has Ser-363 substituted for Tyr-363 and exhibits k,., values for Fru-1,6-P* which are fairly close to that of isozyme B. The general properties of BA306* resembled those of isozyme B. Consequently, these results indicate that Tyr-363 is primarily responsible for exhibiting the higher catalytic activity toward Fru-1,6-P* in isozyme A, but not in isozyme B. This is consistent with the conclusion previously deduced by using the enzyme modified by proteolytic digestion of the C termini (15, 16) and altered isozymes A and B with the Tyr-363 --, Ser-363 substitution generated by site-directed mutagenesis (aldolases A-Y363S and B-Y363S) (17).

Chimeric Enzyme BAB-BAB was essentially indistin- guishable from isozyme B in many respects, i.e. the kcat values for the Fru-1,6-Pn cleaving reaction, the K, for Fru-1,6-P* and Fru-l-P, and the Fru-1,6-P,/Fru-1-P activity ratio. How- ever, the kcat of this chimeric enzyme for Fru-1-P was some- what lower than that of isozyme B. Such characteristics for BAB were plausible because the regions derived from isozyme A (positions 212-306) are highly conserved among vertebrate aldolases A, B, and C. Furthermore, Ser-217, Pro-235, and Gln-241, all of which are conserved in isozyme A, were re- placeable with the corresponding residues of isozyme B (Asn- 217, Ala-235, and Lys-241) with little or no loss in activity (18). Thus, it can be concluded that at least the sequences consisting of CCS-6 and, in part, CCS-5 and CCS-7 play a role in the basal function common to the three isozymic forms.

Chimeric Enzyme BA137-In contrast to the six chimeric enzymes described above, BA137, which was particularly un-

FIG. 4. Thermostability of normal and chimeric human al- dolases. Heat treatment was performed for 30 min at the tempera- tures indicated, and the residual activity was measured in the presence of Fru-1,6-Pz, under the standard assay conditions (see “Materials and Methods”). Results are presented as percent activity of the untreated enzyme.

usual in nature and rather similar to human aldolase A- Y363S, did not exhibit the activities of isozyme A at all (17). That is, its ka, value for Fru-1,6-P* was as low as %S that of isozyme A and i/s that of isozyme B, although the K,,, values for Fru-1,6-Pa and Fru-1-P of this chimeric enzyme were almost comparable to those of isozyme B (Table I). As will be described elsewhere,’ monoclonal antibody 3C5, a monoclonal anti-human aldolase A antibody, interacted with this chimeric enzyme with low efficiency, although it interacted equally well with all other chimeric enzymes and isozyme A. Moreover, BA137 is thermolabile (see Fig. 4). Thus, it appears to be a chimeric enzyme with an unusual conformation resulting in a loss of catalytic activity. Such similar but unusual charac- teristics were also observed with the eighth chimeric enzyme, BA133.3 The factors that endow BA133 and BA137 with such peculiar characteristics remain to be elucidated. The region corresponding to CCS-4 (Fig. 1, region c-C-d) is shown to participate in formation of a p-barrel structure that plays a role in keeping the acidic, basic, and hydrophobic residues in the interior cavity of the p-barrel, which are either directly or indirectly required for catalytic activity, at the right position (11). Furthermore, as they are highly conserved among the three isozymic forms (Fig. l), it is possible that the modifi- cation at the relevant region could impose unfavorable StNC- turelfunction relationships upon the enzyme. This possibility was strongly supported by the results of aldolases from pa- tients with hereditary diseases; aldolase A-D128G, an altered isozyme A from a hemolytic anemia patient with substitution of Gly for Asp at position 128, was shown to have an unusual conformation with low catalytic activity, high thermolability, and low helical content (18, 20). Similarly, isozyme B from patients having hereditary fructose intolerance with substi- tution of Pro for Ala at position 149 showed a catalytic defect (22).

Other Properties Thermostability of chimeric enzymes was compared with

that of normal isozymes A and B. As shown in Fig. 4, the

* Y. Kitajima, Y. Takasaki, I. Takahashi, and K. Hori, manuscript in preparation.

3 Y. Kitajima, Y. Takasaki, I. Takahashi, and K. Hori, unpublished data.

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Chimeric Human Aldolase Isozymes 17497

200 210 220 230 240

Wave length (nm)

FIG. 5. CD spectra of normal and chimeric mutant enzymes. Circular dichroism spectra of isozymes A and B and five different chimeric enzymes were measured with a JASCO 5600 spectrophotom- eter at 25 “C in 0.02 M Tris-HCl, pH 7.5.

temperatures causing enzymes to have half-maximal activity for the Fru-1,6-P* cleavage were 58 “C for isozyme A and 48 “C for isozyme B, which is in accordance with results shown in a previous paper (17). Those of chimeric enzymes BA34, BA212, and BA306 were almost intermediate between those of isozymes A and B, whereas those of the two other chimeric enzymes, BA306* and BAB, were very similar to that of isozyme B. BA108 was also similar to isozyme B. BA137, however, was shown to be extremely thermolabile.

Conformational differences between normal and some chi- merit mutant enzymes were observed at CD spectra below 250 nm (Fig. 5). Isozyme A showed a large negative molecular ellipticity ([e]) around 220 nm. The CD negative troughs around 220 nm for chimeric enzymes BA212, BAB, and BA306* and aldolase A-Y363S were in between those for isozymes A and B. In contrast, BA306 showed a marked change, and the CD negative trough at 220 nm for this chimeric enzyme was much smaller than that for normal isozyme B. Although the primary cause remains unknown, this unexpected structural feature may be related to the catalytic activities of this enzyme toward Fru-1,6-P2 and Fru- l-P, which are twice as high as those of isozyme B.

In this study, we conventionally employed kcat and K,,, values and the Fru-1,6-P*/Fru-1-P activity ratio as the criteria to distinguish isozymes A and B from each other. In spite of the limited numbers of chimeric constructions, we were able to solve some questions concerning the structure/function relationships. (i) For isozyme A, some isozyme group-specific residues or stretches near Lys-107 as well as Lys-107 and the C-terminal Tyr-363 are required for the higher catalytic ac- tivity toward Fru-1,6-Pz. Conversely, the region spanning residues 108-212 is responsible for the lower activity toward Fru-1-P. (ii) For isozyme B, the regions spanning positions 108-212, on which a postulated C-l phosphate-binding site (Lys-146 or Arg-148) resides, are absolutely required to ex- hibit the higher catalytic activity toward Fru-1-P. Further-

more, the more upstream sequence of positions l-107 is responsible for the lower catalytic activity toward Fru-1,6-P*. (iii) At least residues 212-306, a long stretch near the active- site Lys-229 and highly conserved among isozymes A, B, and C, may be required for the basal framework of the aldolase molecule to exhibit the activity common to the three isozymic forms.

In a series of experiments using site-directed mutagenesis (17,18,21), we showed that Lys-107 and Tyr-363 of isozyme A were absolutely required for catalytic activity and Asp-128 for its stability, whereas neither Cys-72 nor Cys-338, which were supposed to be essential for catalysis, participated in the reaction (17, 18). As described above, we also showed that Gln-241 conserved specifically in isozyme A and Ser-217 and Pro-235 conserved in CCS-5 of isozymes A and C were re- placeable with Lys-241, Asn 271, and Ala-235 of isozyme B, respectively, without a significant loss in activity (18). In future studies, a site-directed mutagenesis in conjunction with chimeric enzyme analyses would be required to obtain more precise information on a possible relationship between partic- ular amino acid residue(s) in the molecule and isozyme- specific function.

Acknowledgments-We thank Professor Takeharu Hisatugu for his interest and encouragement during this work. We also thank Drs. Eiji Kimoto and Setsuko Ando (Fukoka University) for performing the CD spectrum analyses and Lisa Tsukamoto for preparation of the manuscript.

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A and B. Analysis of molecular regions which determine isozyme-specific Construction and properties of active chimeric enzymes between human aldolases

1990, 265:17493-17498.J. Biol. Chem. 

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