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PubMed2006

Crystal structure of yeast peroxisomal multifunctional enzyme: structural basis for substrate specificity of (3R)-hydroxyacyl-CoA dehydrogenase units.

(3R)-hydroxyacyl-CoA dehydrogenase is part of multifunctional enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric multifunctional enzyme is very different.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1997

Structure-function studies of p38 mitogen-activated protein kinase. Loop 12 influences substrate specificity and autophosphorylation, but not upstream kinase selection.

Several mitogen-activated protein kinase (MAPK) cascades have been identified in eukaryotic cells. The activation of MAPKs is carried out by distinct MAPK kinases (MEKs or MKKs), and individual MAPKs have different substrate preferences. Here we have examined how amino acid sequences encompassing the dual phosphorylation motif located in the loop 12 linker (L12) between kinase subdomains VII and VIII and the length and amino acid sequence of L12 influence autophosphorylation, substrate specificity, and upstream kinase selectivity for the MAPK p38. Conversion of L12 of p38 to an "ERK-like" structure was accomplished in several ways: (i) by replacing glycine with glutamate in the dual phosphorylation site, (ii) by placing a six-amino acid sequence present in L12 of ERK (but absent in p38) into p38, and (iii) by mutations of amino acid residues in loop 12. Two predominant effects were noted: (i) the Xaa residue in the dual phosphorylation motif Thr-Xaa-Tyr as well as the length of L12 influence p38 substrate specificity, and (ii) the length of L12 plays a major role in controlling autophosphorylation. In contrast, these modifications do not result in any change in the selection of p38 by individual MAPK kinases.

Amino Acid Sequence↗
PubMed2001

Reciprocal activation by cyclin-dependent kinases 2 and 7 is directed by substrate specificity determinants outside the T loop.

Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.

Amino Acid Motifs↗
PubMed2003

Substrate specificity of the nonribosomal peptide synthetase PvdD from Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdine(PAO), which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified. L-Threonine, one of the amino acid residues in pyoverdine(PAO), was an effective substrate for the recombinant protein in ATP-PP(i) exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including D-threonine, L-serine, and L-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases.

Amino Acid Sequence↗
PubMed1982

Substrate specificity of CTP synthetase from Escherichia coli.

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.

Carbon-Nitrogen Ligases↗
PubMed1976

Hepatic 15-hydroxylation of corticosteroids in the rat. Substrate specificity studied in the isolated perfused liver.

The substrate specificity of a sex-specific hepatic 15-hydroxylase active on different C21O2 and C21O3 steroids was studied in the isolated perfused liver from female rats. Liquid-chromatographic separation methods in combination with computerized gas chromatography-mass spectrometry was employed to identify the metabolites formed. The majority (between 75-90%) of 15-hydroxylated compounds isolated were present as monosulphate conjugates while smaller amounts of disulphates were also detected. Hydroxylation was found to take place exclusively at position 15 beta. A certain number of 11-deoxy-21-hydroxy, 11-oxo-21-hydroxy and 11,21-dihydroxy steroids with a 3-keto-delta4-, 3-keto-5 alpha(5 beta)-, 3alpha, 5alpha- or 3 beta, 5 beta-structure were readily converted to 15 beta-hydroxylated metabolites. Depending on the structure of the substrate, between 20 and 87% of the total metabolites formed were 15 beta-hydroxylated. 5 alpha-Reduced steroids were better substrates for the hydroxylase than the corresponding 3-keto-delta4- or 5 beta-reduced compounds. The configuration of the hydroxylgroup at C-3 did not affect the degree of 15-hydroxylation. 11 beta-Hydroxylated steroids served as better substrates than the corresponding 11-dehydro-, 11-deoxy- or 11 alpha-hydroxy compounds. 5 alpha-Dihydrocorticosterone and 3 alpha, 5 alpha-tetrahydrocorticosterone were the best substrates for the 15 beta-hydroxylase.

Animals↗
PubMed1994

Expression, purification, and substrate specificity of isocitrate dehydrogenase from Thermus thermophilus HB8.

Isocitrate dehydrogenase (ICDH) from an extreme thermophile, Thermus thermophilus HB8, was overexpressed in Escherichia coli. The enzyme was easily purified to homogeneity by a combination of heat treatment (70 degrees C, 20 min) and column chromatography. The N-terminal sequence of the protein thus purified coincided with that of the protein extracted from the thermophile. The substrate specificity of the enzyme was mutationally analyzed and engineered to recognize 3-alkyl-malate as a substrate. Based on the three-dimensional structure of E. coli isocitrate dehydrogenase, Ser97 qnd Asn99 of the thermophile enzyme were speculated to participate in the substrate recognition, and these residues were replaced with threonine and leucine, respectively. Molecular recognition of the mutant enzymes, [S97T]ICDH, [N99L]ICDH, and [S97T, N99L]ICDH, were studied using isocitrate, 3-isopropylmalate, and 3-ethylmalate. The affinity toward isocitrate was reduced in the cases of [S97T]ICDH and [N99L]ICDH, confirming the importance of the residues for the reaction. Though none of the mutants acted on 3-isopropylmalate, [N99L]ICDH was competitively inhibited by 3-isopropylmalate with a higher affinity than that of the wild-type enzyme. [N99L]ICDH showed an approximately 10(3)-fold higher value of (kcat/Km)3-ethylmalate/(kcat/Km)isocitrate than the wild-type enzyme, indicating that the single mutation of Asn99 to leucine switched the substrate specificity of the enzyme away from isocitrate and toward 3-ethylmalate.

Amino Acid Sequence↗
PubMed1998

Substrate specificity of retinyl ester hydrolase activity in retinal pigment epithelium.

In the eye, hydrolysis of stored retinyl esters is catalyzed by retinyl ester hydrolase (REH) activities in retinal pigment epithelium (RPE) membranes. In the present study, biochemical analyses were conducted to determine the substrate specificity of these activities. Specific activities determined for hydrolysis of various retinol isomers of retinyl palmitate (9-cis-, 11-cis-, 13-cis-, and all-trans-retinyl palmitates) indicated that 11-cis-retinyl palmitate is preferentially hydrolyzed (1.7 nmol/min/mg) compared to the other isomers (0.1-0.3 nmol/min/mg). Examination of the specificity of REH activity for 11-cis-retinyl esters of varied acyl chain length (-myristate, -palmitate, and -stearate) and degree of saturation (-oleate and -linoleate) further demonstrated that palmitate is the preferred fatty acyl moiety. Notably, retinyl esters possessing chain lengths which more closely approximate that of the palmitate ester exhibited higher rates of hydrolysis. Similar results were obtained in retinyl ester-plasma membrane fusion studies in which hydrolysis took place within the membrane domain rather than at the lipid-water interface. REH substrate specificity was further assessed in competition studies in which 11-cis-retinyl palmitate hydrolysis was monitored in the presence of 13-cis-, 9-cis-, or all-trans-retinyl palmitate. Results show that addition of these retinyl palmitate isomers does not affect the rate of hydrolysis of 11-cis-retinyl palmitate. However, the hydrolytic rates associated with other retinyl palmitate isomers were significantly reduced in the presence of 11-cis-retinyl palmitate. Finally, cholesterol ester hydrolase activity was found to be distinct from the observed 11-cis-REH activity and the presence of cholesterol oleate did not affect the rate of 11-cis-retinyl palmitate hydrolysis. Collectively, these data support the hypothesis that a distinct, membrane-associated, 11-cis-retinyl palmitate-specific retinyl ester hydrolase activity exists in the retinal pigment epithelium.

Animals↗
PubMed2003

Analysis of helicase activity and substrate specificity of Drosophila RECQ5.

RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3' Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5' Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM.

Animals↗
PubMed1991

Deoxynucleoside phosphorylating enzymes in monkey and human tissues show great similarities, while mouse deoxycytidine kinase has a different substrate specificity.

Three key enzymes in the anabolic phosphorylation of deoxyribonucleosides and deoxyribonucleoside analogs were purified i.e. cytoplasmic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and cytoplasmic deoxycytidine kinase (dCK) from human, mouse and monkey liver and spleen. Their subunit structure and substrate specificities were compared. Extensive purification of TK1 and dCK from mouse spleen and TK2 from mouse and monkey livers revealed major polypeptide bands of 25, 30 and 28 kD, respectively, on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which are very similar to the subunit molecular weights of the corresponding human enzymes. Affinity purified polyclonal antibodies against human dCK also cross-reacted with 30 kD bands in extracts from both mouse and monkey spleen. Thus, the molecular weights of the subunits of these three enzymes appeared to be very similar in all three species. TK1 and TK2 from these different sources appeared to have similar substrate specificities against several deoxyribonucleoside analogs. However, mouse dCK differed significantly from monkey and human dCK in its capacity to phosphorylate dAdo and 2',3'-dideoxycytidine (ddCyd) with a Vmax approximately 10-fold lower than that of the two latter enzymes. The Km and Vmax values for dCyd and arabinocytosine appeared to be very similar with the enzymes from all three species. The fact that mouse dCK shows low activity with dAdo and ddCyd explains differences reported previously in the metabolism of dAdo and ddCyd in mouse compared to that in human lymphocytes. These results argue against the use of mice as model systems for human deoxynucleoside metabolism.

Animals↗
PubMed2003

Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities.

Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

Arachis↗
PubMed2004

Polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas stutzeri 1317 had different substrate specificities.

The whole polyhydroxyalkanoate (PHA) synthesis gene locus of Pseudomonas stutzeri strain 1317 containing PHA synthase genes phaC1Ps, phaC2Ps and PHA depolymerase gene phaZPs was cloned using a PCR cloning strategy. The sequence analysis results of the phaC1Ps, phaC2Ps and phaZPs showed high homology to the corresponding pha loci of the known Pseudomonas strains, respectively. PhaC1Ps and PhaC2Ps were functionally expressed in recombinant Escherichia coli strains and their substrate specificity was compared. The results demonstrated that PhaC1Ps and PhaC2Ps from P. stutzeri 1317 had different substrate specificities when expressed in E. coli. In details, PhaC2Ps could incorporate both short-chain-length 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates (mcl 3HA) into PHA, while PhaC1Ps only favored mcl 3HA for polymerization.

Acyltransferases↗
PubMed1997

Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools.

Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible for the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1' of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed at position P1. Position P2' accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3' and P4' were also defined by pool sequencing. Furthermore, pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase.

Amino Acid Sequence↗
PubMed2006

Insights into the substrate specificity of a novel snake venom serine peptidase by molecular modeling.

The cDNA encoding BthaTL, a serine peptidase from the venom of the snake Bothrops alternatus, was cloned and sequenced. The deduced primary structure shows over 62% of identity with snake venom thrombin-like enzymes (SVTLEs), molecules with high substrate specificity toward different natural substrates. Indeed, a phylogenetic reconstruction by two different methods clustered this enzyme close to other SVTLEs. These enzymes generally affect the hemostatic system in several ways, and therefore are used as tools in pharmacology and clinical diagnosis. A three-dimensional model of BthaTL was built by homology modeling using TSV-PA (Trimeresurus stejnegeri venom plasminogen activator) crystal structure as template. BthaTL model showed that the typical catalytic triad conformation of serine peptidases was preserved. The calcium coordination ligands were absent or adopt an unfavorable conformation, preventing interactions with metals. On the other hand, the Asp97-Arg174 saline bridge of TSV-PA was not found and its specificity determinant Phe193 is replaced by a Gly in BthaTL. The substitution of essential residues in the neighborhoods of the catalytic site cleft of BthaTL indicates that these two proteins do not share the same enzymatic specificity, what means that BthaTL will probably not activate plasminogen. Such observations may be helpful in the understanding of the molecular mechanism for substrate specificity of these enzymes.

Amino Acid Sequence↗
PubMed1988

Substrate specificity of duckling hepatic and renal D-amino acid oxidase.

The substrate specificity of duckling hepatic and renal D-amino acid oxidase (DAAO; D-amino acid: O2 oxidoreductase [deaminating], E.C. 1.4.3.3) was determined using a method based on the combination of coupled enzyme reactions and a colorimetric procedure. When activities were averaged across tissues, D-proline was the most reactive substrate, followed by (in order) D-phenylalanine, D-alanine, D-methionine, D-leucine, D-isoleucine, D-valine, D-tryptophan, D-arginine, and D-lysine. Compared with D-alanine, duckling DAAO had minimal or no reactivity with D-asparagine, D-glutamine, D-histidine, D-threonine, D-cysteine, glycine, or D-serine. These results were in general agreement with data from other vertebrate species.

Animals↗
PubMed1992

Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants.

To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain of src or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology.

Amino Acid Sequence↗
PubMed1999

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1981

Substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli.

The investigation of the substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli allows a description of the binding region of pyruvate. Substrate analogs with electronegative substitutions in the methyl group show a strong competitive inhibition of the overall reaction of the pyruvate dehydrogenase complex. The most efficient inhibitor is fluoropyruvate which has a more than 100-fold higher affinity for the enzyme than pyruvate (Ki = 1.4 x 10(-6) M) does. The affinity of alpha-keto acids decreases with increasing chain length. Branched chain alpha-keto acids are even less effective inhibitors (Ki = approximately 0.02 M). alpha-Ketobutyrate is the only alpha-keto acid which is able to substitute for pyruvate as a substrate in the overall reaction of the enzyme complex. The Km value (3 mM) is 10-fold greater than that for pyruvate. The steady state kinetics of the overall reaction of alpha-ketobutyrate exhibits the same cooperativity (nh = 1.9) as seen with pyruvate. Small modifications of the carbonyl or the carboxyl group of pyruvate prevent binding completely. Binding of pyruvate to the pyruvate dehydrogenase complex may thus require interaction with two independent electrophilic centers. The acceptance of the methyl group seems not so much due to lipophilic interactions as to a steric effect. The experiments were carried out with an enzyme which was purified by a modified procedure which is faster and more convenient than previous methods. The procedure is applicable up to 0.5 liter of crude extract.

Binding Sites↗