Characterization of the monomer-dimer equilibrium of human cytomegalovirus protease by kinetic methods

Herpesviruses encode a serine protease that is essential for the maturation of infectious virions. This protease has a unique polypeptide backbone fold and contains a novel Ser-His-His catalytic triad. It exists in a monomer-dimer equilibrium in solution, but only the dimer form of the enzyme is catalytically active. The stability of this dimer is affected by the presence of anti-chaotropic agents. Most of the reported Kd values for this dimer (between 0.6 and 6 microM) are inconsistent with the fact that the protease is routinely assayed at 20-50 nM concentrations, as only monomeric species would be expected with such Kd values. We have characterized the monomer-dimer equilibrium of HCMV protease using a new method, which observes the exchange between dimers of the wild-type enzyme and the active-site Ser132Ala mutant in a titration experiment. The Kd of the dimer was determined to be 8 microM and 31 nM in the absence or presence of anti-chaotropic agents (10% glycerol and 0.5 M Na2SO4), respectively. Detailed kinetic analysis also showed that, in addition to the 260-fold stabilization of the dimer, the anti-chaotropic agents produced a 7-fold enhancement in the catalytic activity of the dimer.     

Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus.

A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.     

Multimeric structure of the secreted meprin A metalloproteinase and characterization of the functional protomer

Meprin A secreted from kidney and intestinal epithelial cells is capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. The secreted form of meprin A is a homo-oligomer composed of alpha subunits, a multidomain protease of 582 amino acids coded for near the major histocompatibility complex of the mouse and human genome. Analyses of the recombinant homo-oligomeric form of mouse meprin A by gel filtration, nondenaturing gel electrophoresis, and cross-linking (with disuccinimidyl suberate or N-(4-azido-2,3,5,6-tetraflourobenzyl)-3-maleimidylpropionamide) indicate that the secreted enzyme forms high molecular weight multimers, with a predominance of decamers. The multimers are composed of disulfide-linked dimers attached noncovalently by interactions involving the meprin, A5 protein, receptor protein-tyrosine phosphatase mu (MAM) domain. The active protomer is the noncovalently linked dimer. Linkage of active protomers by disulfide-bonds results in an oligomer of approximately 900 kDa, which is unique among proteases and distinguishes meprin A as the largest known secreted protease. Electron microscopy revealed that the protein was present in two states, a crescent-shaped structure and a closed ring. It is concluded from this and other data that the covalent attachment of the protomers enables noncovalent associations of the native enzyme to form higher oligomers that are critical for hydrolysis of protein substrates.     

Role of isoprenoid lipids on the heterotrimeric G protein gamma subunit in determining effector activation.

Post-translational prenylation of heterotrimeric G protein gamma subunits is essential for high affinity alpha-beta gamma and alpha-beta gamma-receptor interactions, suggesting that the prenyl group is an important domain in the beta gamma dimer. To determine the role of the prenyl modification in the interaction of beta gamma dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the gamma1, gamma2, and gamma11 subunits were altered to direct modification with different prenyl groups. Six recombinant beta gamma dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-beta isozymes and type II adenylyl cyclase. The native beta1 gamma2 dimer (gamma subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-beta than either the beta1 gamma1 (farnesyl) or the beta1 gamma11 (farnesyl) dimers. However, farnesyl modification of the gamma subunit in the beta1 gamma2 dimer (beta1 gamma2-L71S) caused a decrement in its ability to activate phospholipase C-beta. In contrast, both the beta1 gamma1-S74L (geranylgeranyl) and the beta1 gamma11-S73L (geranylgeranyl) dimers were more active than the native forms. The beta1 gamma2 dimer activates type II adenylyl cyclase about 12-fold; however, neither the beta1 gamma1 nor the beta1 gamma11 dimers activate the enzyme. As was the case with phospholipase C-beta, the beta1gamma2-L71S dimer was less able to activate adenylyl cyclase than the native beta1 gamma2 dimer. Interestingly, neither the beta1 gamma1-S74L nor the beta1 gamma11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the gamma subunit and its prenyl group play a role in determining the activity of the beta gamma-effector complex.     

Methionyl-tRna synthetase from wheat germ. Effect of an endogenous protease and correlations between structural characteristics and catalytic properties

Methionyl-tRNA synthetase (MetRS) has been described as a free monomeric or oligomeric enzyme; or included in a multienzyme complex. Moreover, on limited tryptic digestion, it can generate shorter forms. So, when purified from wheat-germ lysate, the possible presence of proteases able to hydrolyse this enzyme was investigated. When extraction was performed with sulfhydryl-blocking reagents, an active monomeric MetRS of Mr 105,000 was purified. This enzyme form was identical to the structure exhibiting methionyl-tRNA synthetase activity in multienzyme complexes. Without this inhibitor, MetRS was purified as an active dimeric form of Mr 165,000 with identical subunits of Mr 82,000. A protease inhibited by sulfhydryl-blocking reagents and included in a complex of Mr 2.10(6) was isolated from this wheat-germ lysate. This protease was able to hydrolyse different proteins (albumin, casein), but was without activity for a trypsin substrate, such as N-alpha-benzoyl-DL-arginine p-nitroanilide. When added to a solution of Mr-105,000 MetRS, it yielded an inactive peptide of Mr 20,000, containing numerous charged amino acids and a protein of Mr 82,000, able to give an active dimeric enzyme of Mr 165,000. Amino acid analysis of this last form, indicated an identical structure with the active dimeric MetRS of Mr 165,000, purified in the absence of protease inhibitors. Moreover, the affinity for methionine was the same for the monomeric enzyme of Mr 105,000 and the dimeric form of Mr 165,000, probably because proteolysis did not affect the catalytic domain. When enzymic activity of the proteolyzed form (Mr 2 x 82,000) was studied versus enzyme concentration, a decrease in specific activity, at low concentrations, was seen. This phenomenon was analysed on the basis of the existence of an equilibrium between an active dimer and two inactive monomers. With the active monomeric form of Mr 105,000, no change in specific activity with decreasing enzyme concentration occurred.     

Long range effects of amino acid substitutions in the catalytic chain of aspartate transcarbamoylase. Localized replacements in the carboxyl-terminal alpha-helix cause marked alterations in allosteric properties and intersubunit interactions.

A single alpha-helical polypeptide segment of 21 amino acids near the carboxyl terminus of the catalytic chain of aspartate transcarbamoylase from Escherichia coli has been shown recently to be important for the in vivo folding of the chains and assembly of the enzyme (Peterson, C. B., and Schachman, H. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 458-462). Calorimetric measurements on purified mutant enzymes showed that single amino acid replacements within this secondary structural element affect the overall thermal stability of the oligomeric enzyme and the energetics of the interactions between polypeptide chains within the holoenzyme. Studies presented here demonstrate that marked changes in cooperativity occur due to single amino acid substitutions. Replacement of Gln288 by either Ala or Glu leads to a striking increase in the Hill coefficient of the holoenzymes and a substantial increase in the aspartate concentration corresponding to one-half Vmax. In contrast, the isolated catalytic trimers harboring these same substitutions were similar in activity to the wild-type subunit, with the same affinity for aspartate as indicated by the values of Km. Substituting Ala for the only charged residue in the helix, Arg296, caused a marked reduction in enzyme activity, as well as a greatly reduced stability of the holoenzyme due to a substantial weakening of the interactions between the catalytic and regulatory subunits. A subunit exchange method was used to demonstrate the changes in interchain interactions resulting from the amino acid substitutions and to show the additional weakening upon the binding of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, at the active sites. Taken together, the results on this series of mutant enzymes illustrate how the effects of single amino acid replacements in one element of secondary structure are propagated throughout the molecule to positions remote from the site of the substitution.     

Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site

BACKGROUND: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. RESULTS: We have determined the crystal structure of cyanase at 1.65 A resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. CONCLUSIONS: The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding alpha-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed.     

Protein phosphatase type 1 catalytic subunit forms nondissociable dimers

Protein phosphatase type 1 is the major enzyme in skeletal muscle and liver for the dephosphorylation of Ser(P) and Thr(P) phosphoproteins. The cDNA for the catalytic subunit encodes a polypeptide of Mr 35,400 kDa, consistent with the Mr of 36,000-38,000 of the active protein purified in various laboratories. However, several investigators have found a Mr 70,000 protein for phosphatase type 1. In this report proteins of Mr 38,000 and 70,000 were resolved by Mono Q chromatography after extensive copurification from rabbit skeletal muscle. Antibodies affinity-purified against a type 1 phosphatase catalytic fragment reacted with both proteins in Western immunoblotting. Fractions from each peak were cleaved with cyanogen bromide and the major peptides were the same size by electrophoresis in gradient polyacrylamide gels. Cyanogen bromide peptides of the individual bands also were mapped by reversed-phase high-performance liquid chromatography. The purified Mr 38,000 and 70,000 proteins had identical HPLC peptide maps and also gave the same amino acid compositions after acid hydrolysis. Purified Mr 38,000 phosphatase catalytic subunit spontaneously formed a Mr 70,000 dimer that resisted usual dissociation conditions, i.e., boiling dodecyl sulfate plus 2-mercaptoethanol, but could be cleaved to about half size by various proteases, indicating that monomers were bound together near their amino or carboxy termini. Physiological changes in protein phosphatase type 1 are reflected in the amount of nondissociable dimers detected in tissue extracts.     

Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues

Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.     

Purification and characterization of thiol aminopeptidase from the cytosolic fraction of human placenta

A thiol-dependent aminopeptidase was purified from the cytosolic fraction of human placenta. The purified enzyme consisted of a single polypeptide chain with a mol wt of 95,000. The enzyme was most active in the neutral region with Ala-pNA as substrate, and the activity was increased about 20-fold in the presence of some -SH compounds. The results of substrate specificity studies indicated that the enzyme hydrolyzes bonds involving the amino groups of neutral and basic amino acid residues. However, higher thiol-dependent activity was only detected with neutral ones. The enzyme was strongly inhibited by microbial aminopeptidase inhibitors, puromycin, o-phenanthroline, and sulfhydryl reactive-reagents. As to several naturally occurring peptides tested, the enzyme showed N-terminal Tyr-releasing activity toward enkephalins and kinin-converting activity.     

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.     

Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays).

A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.     

Purification of rat liver nuclear protein kinase NI.

Rat liver nuclear protein kinase NI, which appears in the flowthrough of DEAE-Sephadex columns, has been purified approximately 15,000-fold from soluble nuclear protein with yields of up to 10%. The method of purification involved chromatography of the DEAE-flowthrough protein successively on phosvitin-Sepharose and casein-Sepharose followed by rechromatography on phosvitin-Sepharose. The purified enzyme has an s20,w and molecular weight of 3.7 and 47,000, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. A similar molecular weight of 42,000 was determined by gel filtration using Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide with a molecular weight of 25,000. Protein kinase NI therefore consists of a dimer of two identical subunits. Protein kinase NI exhibits maximal activity on casein substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP when either casein or histone H2b is used as a substrate.     

Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.     

The structure of 4-hydroxybenzoyl-CoA thioesterase from arthrobacter sp. strain SU

The 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a thioesterase. Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU. The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called "hot dog fold" composed of six strands of anti-parallel beta-sheet flanked on one side by a rather long alpha-helix. The dimers come together to form the tetramer with their alpha-helices facing outwards. This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their alpha-helices projecting toward the interfacial region. In the Arthrobacter thioesterase, each of the four active sites is formed by three of the subunits of the tetramer. On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter thioesterase. Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme. Although these two bacterial thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Structural characteristics of prostaglandin synthetase from sheep vesicular gland

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.     

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.     

Allosteric communication network in the tetrameric restriction endonuclease Bse634I

Restriction endonuclease Bse634I is a homotetramer arranged as a dimer of two primary dimers. Bse634I displays its maximum catalytic efficiency upon binding of two copies of cognate DNA, one per each primary dimer. The catalytic activity of Bse634I on a single DNA copy is down-regulated due to the cross-talking interactions between the primary dimers. The mechanism of signal propagation between the individual active sites of Bse634I remains unclear. To identify communication pathways involved in the catalytic activity regulation of Bse634I tetramer we mutated a selected set of amino acid residues at the dimer-dimer interface and analysed the oligomeric state and catalytic properties of the mutant proteins. We demonstrate that alanine replacement of N262 and V263 residues located in the loop at the tetramerisation interface did not inhibit tetramer assembly but dramatically altered the catalytic properties of Bse634I despite of the distal location from the active site. Kinetic analysis using cognate hairpin oligonucleotide and one and two-site plasmids as substrates allowed us to identify two types of communication signals propagated through the dimer-dimer interface in the Bse634I tetramer: the inhibitory, or "stopper" and the activating, or "sync" signal. We suggest that the interplay between the two signals determines the catalytic and regulatory properties of the Bse634I and mutant proteins.