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PubMed1991

Distinct developmental regulatory mechanisms for two members of the aldolase gene family.

The aldolase isozyme family is composed of three members, A, B, and C, which are encoded by separate genes. The proteins are expressed in a tissue-restricted manner during development and in the adult. To elucidate the regulation of aldolase mRNA in the mouse liver, we analyzed its expression by a number of methods including Northern blot, RNA dot blot, and nuclear run-on assays. Our experiments demonstrate that the expression of aldolase A in the liver is primarily regulated by post-transcriptional control. In contrast, we found that changes in the level of aldolase B mRNA are due to changes in the rate of initiation of transcription. In addition, we examined the regulation of aldolase expression in the adult kidney. We found that although the kidney has eight times more aldolase B than the liver, the rate of initiation of transcription is similar in both tissues. Also, the rate of initiation of transcription of aldolase A is the same in the adult kidney and liver although there is 40 times more steady state aldolase A mRNA in the kidney than in the liver.

Animals↗
PubMed1998

Hormonal regulation of aldolase B gene expression in rat primary cultured hepatocytes.

Gene expression of aldolase B, an important enzyme for glucose and fructose metabolism, is regulated by hormones. We examined direct effects of major hormones on aldolase B gene expression in rat primary cultured hepatocytes, in comparison with those on the gene expression of phospho(enol)pyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis. Insulin, dexamethasone, and high concentration of glucose increased aldolase B mRNA abundance in the hepatocytes. Glucagon strongly suppressed aldolase B gene expression, and this hormone canceled the stimulative effects of insulin, dexamethasone, and high concentration of glucose. Epinephrine and thyroxine slightly reduced aldolase B mRNA abundance, but these hormones did not cancel the stimulative effects of insulin and dexamethasone. To the contrary, expression of PEPCK gene was suppressed by insulin, dexamethasone, and high concentration of glucose, and remarkably induced by glucagon. Glucagon rapidly suppressed aldolase B gene expression at the transcriptional level. Forskolin and dibutyryl cAMP mimicked the suppressive effect of glucagon on aldolase B gene expression. These results suggest that glucagon may be a key regulator of aldolase B gene transcription through a cAMP/protein kinase A-signaling pathway.

Animals↗
PubMed1976

The differentiation of proximal and distal tubules in the male rat kidney: the appearance of aldolase isozymes, aminopeptidase and alkaline phosphatase during ontogeny.

1. The specific activities of aminopeptidase, alkaline phosphatase and aldolase isozymes were measured in homogenates of kidneys taken at different stages of ontogeny. The cellular localization of these enzymes was studied in cryostat tissue sections using substrate linked assays for aminopeptidase and alkaline phosphatase and the mixed aggregation immuno-cytochemical technique for aldolase isozymes; local enzyme concentrations were estimated photometrically. 2. The presence of both aldolase-A and aldolase-B was demonstrated in all metanephrogenic cells (and at still higher concentrations in collecting tubule cells) of the rat fetus 16 days after conception and in the undifferentiated cells of the neogenetic zone of kidney up to 8 days after birth; no aminopeptidase or alkaline phosphatase could be found in these cells. 3. Measurements made on stained tissue sections show that the shift towards aldolase-B, seen in homogenate analyses, is due to a change in the relative amounts of proximal tubules. No evidence was seen for repression in the synthesis of aldolase-A or aldolase-B monomers in the different kidney cells during ontogeny. 4. Two transitions in the mode of nephron differentiation were observed: one was shortly after birth, the other followed weaning. Before the first transition the concentrations of the enzymes increased to different degrees, such that the enzymes reached concentrations comparable with those as in the cells of adult rats by 2 to 4 days post partum. After the second transition proximal tubule size and specific activity of brush border membrane enzymes increased 3 fold. In contrast, the distal tubules did not increase significantly in size, but their aldolase-A concentration increased 3 fold. 5. Evidence based on enzyme quantification and morphometry in kidney sections is presented to demonstrate that the proximal tubule cells show functional adaptation by two independent mechanisms: specific amplification of gene expression and hypertrophy. In contrast, the distal tubule shows functional adaptation only by specific amplification of gene expression.

Alkaline Phosphatase↗
PubMed1990

Transcriptional regulation of an aldolase gene in the regenerating rat liver.

The relative abundance of rat aldolase A, B, C, alpha-fetoprotein, and albumin mRNAs was determined by Northern hybridization during liver regeneration after partial hepatectomy. Aldolase A mRNA increased more than 10-fold on the 3rd day after partial resection compared with that of normal adult rat liver. S1 analysis revealed that three species of aldolase A mRNAs (mRNA I, II & III) reappeared. However, transcriptional rate of aldolase A mRNA did not change at all during the regeneration. In contrast, aldolase B, aldolase C and albumin mRNAs did not change at all. These findings suggest that the differentiated hepatocyte maintains a differentiated state during the liver regeneration as seen in aldolase B, whereas "oncofetal" isozymes such as aldolase A resurge after partial hepatectomy under the control of post-transcriptional mechanism.

Albumins↗
PubMed1993

Aldolase C in neuroendocrine tumors: an immunohistochemical study.

The expression of cerebral type aldolase C was investigated immunohistochemically in six varieties of neuroendocrine (n = 57) and six types of non-endocrine tumor (n = 76) using the avidin-biotin complex method. Aldolase C expression in the neuroendocrine tumors was also compared with those of chromogranin and gamma enolase. Aldolase C was detected in all the islet cell (7/7) and carcinoid tumors (10/10), thyroid medullary carcinomas (7/7), and pheochromocytomas (10/10), as well as in the majority of neuronal tumors (8/10) and bronchial small cell carcinomas (10/13). Chromogranin immunoreactivity was restricted to the tumors with abundant neuroendocrine granules. Gamma enolase positivity was generally similar to that of aldolase C, but there were some differences. Amongst the bronchial small cell carcinomas, three tumors negative for gamma enolase were positive for aldolase C, while another three tumors were positive for gamma enolase only. However all the small cell carcinomas were positive for at least one of these two enzymes. Aldolase C was detected in 28 (37%) of the 76 non-endocrine tumors and tended to be expressed preferentially in the differentiated portions of these tumors. Although aldolase C was expressed in many bronchial squamous cell carcinomas, the immunoreactivity was localized mainly in keratinizing foci and the less differentiated parts of these tumors expressed the enzyme only occasionally. Thus aldolase C, in conjunction with other neuroendocrine-associated markers, may be of value in identifying tumors of neuroendocrine type.

Adenoma, Islet Cell↗
PubMed2006

Characterization of a Mn-dependent fructose-1,6-bisphosphate aldolase in Deinococcus radiodurans.

The key enzyme of the glycolytic pathway of Deinococcus radiodurans, fructose-1,6-bisphosphate aldolase, could be induced independently by glucose and Mn. The enzyme exhibited the characteristics of the metal-dependent Class II aldolases. Unlike most Class II aldolases, the deinococcal aldolase preferred Mn, not Zn, as a cofactor. The fbaA gene encoding the deinococcal aldolase was cloned and the protein overproduced in various Escherichia coli expression hosts. However, the overexpressed deinococcal enzyme aggregated and formed inclusion bodies. Dissolving these inclusion bodies by urea and subsequent purification by nickel affinity chromatography, resulted in a protein fraction that exhibited aldolase activity only in the presence of Mn. This active aldolase fraction exhibited masses of about 70 kDa and 35 kDa by gel filtration and by SDS gel electrophoresis, respectively, suggesting that the active aldolase was a dimer.

Chromatography, Gel↗
PubMed1983

Endocytosis and degradation of native, cathepsin D-degraded, and glutathione-inactivated aldolase by perfused rat liver.

The uptake and degradation of 125I-labeled (a) native aldolase, (b) cathepsin D-inactivated aldolase, and (c) aldolase inactivated by oxidized glutathione were studied in perfused rat liver. All three forms of aldolase were removed from the perfusion medium and degraded by the liver, but the uptake of the glutathione-inactivated enzyme (half-life in perfusate = 10 min) was much faster than that of the native enzyme (half-life = 30 min) or the cathepsin-inactivated enzyme (half-life = 42 min). The degradation of the enzyme was almost totally inhibited by leupeptin, indicating that thiol proteinases in lysosomes play an important role in the digestion process. Degradation of native and cathepsin D-inactivated aldolase appeared to be slower than that of the glutathione-inactivated enzyme but studies in which liver was preloaded with aldolase by perfusion at 19 degrees C and then warming to 37 degrees C indicated that the rate of degradation of all three forms was similar. It is concluded that the liver is capable of distinguishing between the glutathione-altered aldolase and native or partially degraded aldolase with regard to endocytosis, but that all three forms are degraded at similar rates once within lysosomes.

Animals↗
PubMed1988

Anaerobically regulated aldolase gene of maize. A chimaeric origin?

The sequence of the anaerobically induced fructose 1,6-bisphosphate aldolase gene of maize is presented. Analysis of the upstream sequences of the aldolase gene reveals a six base-pair sequence (TGGTTT) with perfect homology to one of the sub-regions of the anaerobic regulatory element (ARE) which is responsible for the anaerobic induction of the maize alcohol dehydrogenase 1 gene (Adh1). In the aldolase gene this sequence is located at position -70 relative to the start of transcription, in a small segment proven by functional analysis to be important for expression of the aldolase gene. Since this six base-pair sequence has been shown to be critical for anaerobic induction of the Adh1 mRNA, is in the functional promoter region of aldolase and is also present in a homologous position in Adh2 (another anaerobically-induced gene), we suggest this hexanucleotide is essential for anaerobic regulation of each of these genes. The maize aldolase gene is about 50% homologous at the amino acid level to the animal aldolase gene but has a completely different intron/exon structure. While the rat aldolase gene has nine introns the maize gene has a single large intron near the N terminus of the coding region. Because there is 55% homology downstream from the intron and very little homology upstream, we suggest that the maize gene has acquired a 5' region containing signals for anaerobic regulation and fortuitously adding a new N-terminal region to the protein. We must suppose that the plant gene has lost the remaining introns.

Anaerobiosis↗
PubMed1986

Specific, limited tryptic modification of wheat-germ fructose-bisphosphate aldolase subunits: destruction of catalytic activity but not of ability to establish precise subunit-subunit recognition.

We have been using the glycolytic enzyme fructose-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) as a model system to investigate the assembly of oligomeric enzymes. In the present work, we investigate the effect of specific, limited tryptic modification on the properties of aldolase isolated from wheat germ. The wheat-germ enzyme was selected, since several aldolases isolated from animal sources were not readily susceptible to the specific tryptic modification seen with this plant enzyme. We will show that: Low levels of trypsin cause a first-order inactivation of wheat-germ aldolase activity which is associated with a fairly specific cleavage of the enzyme which reduces its subunit molecular weight from 41000 to 39000. The proteolytic modification is greatly inhibited in the presence of the aldolase substrate, fructose bisphosphate. The intact and modified enzymes appear to have similar surface changes, as judged by their behavior during electrophoresis in polyacrylamide gels under non-denaturing conditions. The modified aldolase is not specifically eluted from phosphocellulose columns by fructose bisphosphate under the conditions used in the affinity chromatographic isolation of the intact enzyme, suggesting that the modified enzyme may no longer be able to bind substrate. Although enzymatically inactive, the modified aldolase subunits are able to refold and reassociate into tetrameric combinations following unfolding of the subunits by treatment at low pH; thus, this specific proteolytic modification does not interfere with the ability of wheat-germ aldolase subunits to refold and to establish precise subunit-subunit recognition in vitro.

Carboxypeptidases↗
PubMed2002

Purification and characterization of fructose bisphosphate aldolase from the ground squirrel, Spermophilus lateralis: enzyme role in mammalian hibernation.

Fructose-1,6-bisphosphate (F1,6P(2)) aldolase was purified to homogeneity from skeletal muscle of the golden-mantled ground squirrel, Spermophilus lateralis. Enzyme properties were examined at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C); parallel studies assessed rabbit muscle aldolase for comparison. Kinetic properties of each enzyme were differentially affected by assay temperature. For example, the K(m) for F1,6P(2) of ground squirrel aldolase was 0.9+/-0.05 microM at 37 degrees C and 50% higher (1.45+/-0.04 microM) at 5 degrees C, whereas the K(m) of rabbit aldolase increased threefold over the same temperature range. The inhibitory effects of adenylates were similar at both temperatures for the ground squirrel enzyme, but inhibition by adenosine 5(')-diphosphate, adenosine 5(')-monophosphate, and inosine 5(')-monophosphate was substantially reduced at 5 degrees C for rabbit aldolase. Inhibition by inorganic phosphate increased at lower temperatures for both enzymes; for ground squirrel aldolase, the K(i) was 1.18+/-0.1mM at 37 degrees C and 0.23+/-0.05 mM at 5 degrees C. Inhibition of aldolase by inorganic phosphate could be one factor that helps to shut down glycolysis during hibernation. Thus, mammalian hibernators may exploit low-temperature characteristics of aldolase to benefit the metabolic needs of the hibernating state.

Adenosine Diphosphate↗
PubMed1999

Brownian dynamics simulations of interactions between aldolase and G- or F-actin.

Compartmentation of proteins in cells is important to proper cell function. Interactions of F-actin and glycolytic enzymes is one mechanism by which glycolytic enzymes can compartment. Brownian dynamics (BD) simulations of the binding of the muscle form of the glycolytic enzyme fructose-1,6-bisphosphate aldolase (aldolase) to F- or G-actin provide first-encounter snapshots of these interactions. Using x-ray structures of aldolase, G-actin, and three-dimensional models of F-actin, the electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations provided solution complexes of aldolase with F- or G-actin. All complexes demonstrate the close contacts between oppositely charged regions of the protein surfaces. Positively charged surface regions of aldolase (residues Lys 13, 27, 288, 293, and 341 and Arg 257) are attracted to the negatively charged amino terminus (Asp 1 and Glu 2 and 4) and other patches (Asp 24, 25, and 363 and Glu 361, 364, 99, and 100) of actin subunits. According to BD results, the most important factor for aldolase binding to actin is the quaternary structure of aldolase and actin. Two pairs of adjacent aldolase subunits greatly add to the positive electrostatic potential of each other creating a region of attraction for the negatively charged subdomain 1 of the actin subunit that is exposed to solvent in the quaternary F-actin structure.

Actins↗
PubMed1999

Identification of conserved promoter elements for aldB and isozyme specific residues in aldolase B.

The comparison of three complete aldolase B genes-including known and putative regulatory elements-is presented. The third aldolase B gene was provided by the complete aldB gene sequence (14803 bp) encoding the rabbit aldolase B isozyme. The promoter sequence alignment included the nonmammalian chicken aldolase B gene and confirms the promoter sequence conservation of those elements where trans-factor binding has been demonstrated in rat aldB. Moreover, the alignment reveals conserved sequences that may represent previously unidentified promoter elements that are present in all aldBs or specifically in the mammalian aldB promoters. One remarkable feature is a poly-purine segment found between the CAAT and TATA elements. In the mammalian promoters, this is exclusively a 9-10 bp poly-dA stretch. The avian promoter has an additional stretch of eight dG-bases immediately upstream of the poly-dA. Alignment of a portion of intron 1 of the chicken, human, and rabbit aldB genes reveals conserved sequences that are likely candidates for a reported positive activation sequence. In addition, the amino acid sequences of all eight known aldolase B isozymes is compared to the other vertebrate aldolases. A number of aldolase B-specific residues are identified that cluster in the carboxyl-portion of the sequence. With the exception of residue C268, these residues are not found near the active site, although, they are likely to be responsible for the substrate specificity of aldolase B.

Amino Acid Sequence↗
PubMed1984

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes.

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.

Animals↗
PubMed2001

Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate.

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.

Animals↗
PubMed1988

Degradation of native and modified forms of fructose-bisphosphate aldolase microinjected into HeLa cells.

The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system.

Cathepsin B↗
PubMed1990

Purification and characterization of cytosolic aldolase from carrot storage root.

A single fructose-1,6-bisphosphate (FBP) aldolase has been detected in extracts from carrot storage roots (Daucus carota L.). The enzyme was purified 850-fold to electrophoretic homogeneity and a final specific activity of 26.3 mumols of FBP utilized/min per mg of protein. SDS/PAGE of the final preparation revealed a single protein-staining band of 40 kDa. The native molecular mass was determined by analytical gel filtration to be 159 kDa, indicating that the enzyme is a homotetramer. Denaturing isoelectric focusing revealed two predominant protein-staining bands, with pI values of 5.6 and 5.7. The enzyme is a class I aldolase, since EDTA or metal ions had no effect on its activity. The enzyme was relatively heat-stable, had an activation energy (Ea) of 68.3 kJ.mol-1, and had an absorption coefficient of 8.08 x 10(4) M-1.cm-1 at 280 nm. Km values for FBP and sedoheptulose 1,7-bisphosphate (SBP) were both determined to be 6 microM (pH optima 7.4). The specificity constant with FBP was 2.6 times that obtained with SBP. Ribose 5-phosphate, 6-phosphogluconate, MgAMP, glucose 1-phosphate and phosphoenolpyruvate (PEP) were inhibitors. PEP was a mixed-type inhibitor with respect to FBP (Ki = 3.2 mM, K'i = 5.1 mM). No activators were found. Rabbit anti-(carrot aldolase) polyclonal antibodies immunoprecipitated the activity of both carrot root aldolase and spinach leaf cytosolic aldolase, but not that of spinach leaf plastid aldolase. Western-blot analysis also revealed cross-reactivity with cytosolic, but not plastid, spinach leaf aldolase, indicating that the single carrot root aldolase is cytosolic.

Amino Acids↗
PubMed1998

Genomic sequences of aldolase C (Zebrin II) direct lacZ expression exclusively in non-neuronal cells of transgenic mice.

Aldolase C is regarded as the brain-specific form of fructose-1, 6-bisphosphate aldolase whereas aldolase A is regarded as muscle-specific. In situ hybridization of mouse central nervous system using isozyme-specific probes revealed that aldolase A and C are expressed in complementary cell types. With the exception of cerebellar Purkinje cells, aldolase A mRNA is found in neurons; aldolase C message is detected in astrocytes, some cells of the pia mater, and Purkinje cells. We isolated aldolase C genomic clones that span the entire protein coding region from 1.5 kb 5' to the transcription start site to 0.5 kb 3' to the end of the last exon. The bacterial gene, lacZ, was inserted in two different locations and the constructs tested in transgenic mice. When the protein coding sequences were replaced with lacZ, three of five transgenic lines expressed beta-galactosidase only in cells of the pia mater; one line also expressed in astrocyte-like cells. When lacZ was inserted into the final exon (and all structural gene sequences were retained) transgene expression was observed in astrocytes in all regions of the central nervous system as well as in pial cells. Thus, with the exception of Purkinje cell expression, the behavior of the full-length transgene mimics the endogenous aldolase C gene. The results with the shorter transgene suggest that additional enhancer elements exist within the intragenic sequences. The absence of Purkinje cell staining suggests that the cis elements required for this expression must be located outside of the sequences used in this study.

Animals↗
PubMed2003

Spatial clustering of isozyme-specific residues reveals unlikely determinants of isozyme specificity in fructose-1,6-bisphosphate aldolase.

Vertebrate fructose-1,6-bisphosphate aldolase exists as three isozymes (A, B, and C) that demonstrate kinetic properties that are consistent with their physiological role and tissue-specific expression. The isozymes demonstrate specific substrate cleavage efficiencies along with differences in the ability to interact with other proteins; however, it is unknown how these differences are conferred. An alignment of 21 known vertebrate aldolase sequences was used to identify all of the amino acids that are specific to each isozyme, or isozyme-specific residues (ISRs). The location of ISRs on the tertiary and quaternary structures of aldolase reveals that ISRs are found largely on the surface (24 out of 27) and are all outside of hydrogen bonding distance to any active site residue. Moreover, ISRs cluster into two patches on the surface of aldolase with one of these patches, the terminal surface patch, overlapping with the actin-binding site of aldolase A and overlapping an area of higher than average temperature factors derived from the x-ray crystal structures of the isozymes. The other patch, the distal surface patch, comprises an area with a different electrostatic surface potential when comparing isozymes. Despite their location distal to the active site, swapping ISRs between aldolase A and B by multiple site mutagenesis on recombinant expression plasmids is sufficient to convert the kinetic properties of aldolase A to those of aldolase B. This implies that ISRs influence catalysis via changes that alter the structure of the active site from a distance or via changes that alter the interaction of the mobile C-terminal portion with the active site. The methods used in the identification and analysis of ISRs discussed here can be applied to other protein families to reveal functionally relevant residue clusters not accessible by conventional primary sequence alignment methods.

Amino Acid Sequence↗