Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.     

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.     

SARS CoV main proteinase: The monomer-dimer equilibrium dissociation constant

The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, KD, have varied more than 65,0000-fold, from <1 nM to more than 200 microM. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded KD values of 5.8 +/- 0.8 microM (obtained from the entire scattering curve), 6.5 +/- 2.2 microM (obtained from the radii of gyration), and 6.8 +/- 1.5 microM (obtained from the forward scattering). The KD from chemical cross-linking was 12.7 +/- 1.1 microM, and from enzyme kinetics, it was 5.2 +/- 0.4 microM. While each of these three techniques can present different, potential limitations, they all yielded similar KD values.     

Probing receptor binding activity of interleukin-8 dimer using a disulfide trap

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.     

Kinetics, inhibition and oligomerization of Epstein-Barr virus protease

Epstein-Barr virus (EBV) is an omnipresent human virus causing infectious mononucleosis and EBV associated cancers. Its protease is a possible target for antiviral therapy. We studied its dimerization and enzyme kinetics with two enzyme assays based either on the release of paranitroaniline or 7-amino-4-methylcoumarin from labeled pentapeptide (Ac-KLVQA) substrates. The protease is in a monomer-dimer equilibrium where only dimers are active. In absence of citrate the K(d) is 20 microM and drops to 0.2 microM in presence of 0.5M citrate. Citrate increases additionally the activity of the catalytic sites. The inhibitory constants of different substrate derived peptides and alpha-keto-amide based inhibitors, which have at best a K(i) of 4 microM, have also been evaluated.     

Physicochemical properties of isocitrate dehydrogenase from lactating bovine mammary gland: effect of substrates and cofactors

The influence of substrates and cofactors on the oligomeric structure of the cytosolic form of NADP+-specific isocitrate dehydrogenase (IDH) from lactating bovine mammary gland was investigated using analytical ultracentrifugation and kinetic methods. In guanidine-HCl, the monomer molecular weight for reduced and carboxymethylated IDH was found to be 50,000 to 52,000. In nondenaturing solvents IDH behaves as a homogeneous solute with a molecular weight of 97,200. When added separately, manganous isocitrate, isocitrate, manganous citrate (substrate analog), and a mixture of the substrate analog and NADP+ do not significantly alter the sedimentation coefficient or the molecular weight of IDH as judged by direct observation of the enzyme at 0.1 to 3 microM using sedimentation velocity and equilibrium. Active enzyme sedimentation (AES) was used to assess the degree of dissociation of IDH at lower concentrations, and Kd for the dimer-monomer equilibrium was estimated to be 2 nM. In enzymatic studies, the specific activity at several levels of substrate does not vary as the subunit concentration of enzyme is reduced from 10 to 0.3 nM. Estimates for Kd by AES indicate the presence of a significant fraction of monomer at assay concentrations of 1 nM and below, where the weight fraction of monomer is predicted to be 0.6. If the monomer has a lower activity than the dimer, a drop in specific activity is expected below 1 nM. Significant decreases occur only when the IDH is not protected from denaturation. The concentration of cytoplasmic IDH in bovine mammary tissue is estimated to be 5.7 microM, at least 100-fold greater than our estimates of Kd. Since over 90% of the enzyme is present in the dimeric form, ligand-induced changes in aggregation state cannot play a significant role in the regulation of the cytosolic form of IDH in situ in this tissue.     

8-(2-Carboxymethylthio)-cGMP, a site-1-selective compound for cGMP-dependent protein kinase

The cGMP analogue 8-(2-carboxymethylthio)-cGMP (CMT-cGMP) was synthesized and its binding to cGMP-dependent protein kinase (cGMP kinase) was studied. CMT-cGMP bound at 4 degrees C with an over 1400-fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 microM, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 microM CMT-cGMP at 4 degrees C. The apparent site 1 selectivity was 250-fold at 30 degrees C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 microM for site 1 and 2, respectively. CMT-cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Ka values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 microM CMT-cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT-cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT-cGMP and cGMP inhibited cAMP-stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 microM and 2 microM, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself. These results indicate that CMT-cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expression of a partial enzyme activity.     

Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide

Human immunodeficiency virus 1 (HIV-1) protease is an aspartyl protease composed of two identical protomers linked by a four-stranded antiparallel beta-sheet consisting of the NH2- and COOH-terminal segments (Weber, I.T. (1990) J. Biol. Chem. 265, 10492-10496). Kinetic analysis of the HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate demonstrates that the enzyme is an obligatory dimer. At pH = 5.0, 0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA buffer, 37 degrees C, the equilibrium dissociation constant, Kd = 3.6 +/- 1.9 nM. We found that the tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, corresponding to the COOH-terminal segment of the enzyme, is an excellent inhibitor of the enzyme. Kinetic analysis shows that the inhibitor binds to the inactive protomers and prevents their association into the active dimer (dissociative inhibition). The dissociative nature of this inhibition is consistent with the results obtained from sedimentation equilibrium experiments in which the apparent molecular weight of the enzyme was observed to be 20,800 +/- 1,500 and 12,100 +/- 300, in the absence and presence of the COOH-terminal tetrapeptide, respectively. The dissociation constant of the protomer-inhibitor complex is Ki = 45.1 +/- 1.8 microM. This is the first kinetic analysis and direct experimental demonstration of noncovalent dissociative inhibition.     

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

Analysis of high-affinity assembly for AMPA receptor amino-terminal domains

Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer-dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer-dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer-dimer Kd was 5.6 μM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 μM was less than twofold different from the SV value. Analysis of cell contents after the ～1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.     

Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.     

Effectors of HIV-1 protease peptidolytic activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.     

What is the role of the second “structural” NADP+‐binding site in human glucose 6‐phosphate dehydrogenase?

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.     

Potent inhibition of mammalian progesterone synthesis by 2 alpha-cyanoprogesterone.

2 alpha-Cyanoprogesterone potently inhibits the conversion of [3H]pregnenolone into progesterone catalysed by bovine corpora lutea, bovine adrenal cortex and human term placenta microsomes (microsomal fractions), yielding IC50 (concentration causing 50% inhibition) values of 66 nM, 120 nM and 700 nM respectively. By contrast, it is an exceedingly poor inhibitor of the isomerization of pregn-5-ene-3,20-dione, yielding IC50 values between 50 and 70 microM. On this basis, 2 alpha-cyanoprogesterone would appear to be an extraordinarily selective inhibitor of the 3 beta-hydroxysteroid dehydrogenase. Dixon plots indicate that it is a very-tight-binding competitive inhibitor of the corpus-luteum enzyme, yielding a Ki of 15 nM. In the bovine adrenal cortex and human placenta the steroid is less potent and inhibits the dehydrogenase non-competitively with Ki values of 150 nM and 1.0 microM respectively. Thus 2 alpha-cyanoprogesterone inhibits the corpus-luteum dehydrogenase with substantial selectivity. Because of its high affinity for the ovarian enzyme, the presence of low-micromolar concentrations of 2 alpha-cyanoprogesterone can promote a complete cessation of progesterone synthesis in corpora-lutea microsomes for several hours. Since this effect is observed in the presence of saturating concentrations of pregnenolone (50 microM), it is predicted that this inhibitor may be even more potent in vivo. 2 alpha-Cyanoprogesterone displays very low affinity for the human progesterone receptor, yielding a Kd of 600 nM as against a Kd of 1.6 nM for progesterone. It is suggested that 2 alpha-cyanoprogesterone may be a selective inhibitor of ovarian progesterone synthesis and may act as an effective anti-gestational agent in vivo.     

Dissociation and catalysis in yeast hexokinase A.

1. The specific activity of yeast hexokinase A depends on the concentration of the protein in the solution being assayed. When a solution containing 13.5 mg of hexokinase A/ml is diluted 10--100-fold at various values of pH and temperature, there is a gradual decline in the specific activity of the enzyme until an equilibrium value is reached, which varies with the chosen experimental conditions. 2. The catalytic activity lost when hexokinase A (1 mg/ml) is incubated at 30degreesC is recovered by lowering the temperature to 25degreesC. 3. These concentration- and temperature-dependent phenomena are consistent with the existence of a monomer-dimer equilibrium in which the dimer alone is the catalytic form of the enzyme. 4. Glucose alone prevents the decline in specific activity of hexokinase A after dilution, but it does not re-activate dilute solutions solutions of the enzyme. It is concluded that glucose binds to both the dimer and the monomer and prevents both association and dissociation. 5. The progress curve describing the phosphorylation of glucose catalysed by hexokinase A does not attain a steady state. It is possible that dissociation of catalytically active dimers in a ternary complex with glucose and ATP (or glucose 6-phosphate and ADP) could explain the non-linearity of this progress curve.     

Measurement of the second osmotic virial coefficient for protein solutions exhibiting monomer-dimer equilibrium

The second osmotic virial coefficient (B) is a measure of solution nonideality that is useful for predicting conditions favorable for protein crystallization and for inhibition of aggregation. Static light scattering is the technique most commonly used to determine B values, typically using protein concentrations less than 5 mg/mL. During static light scattering experiments at low protein concentrations, frequently the protein is assumed to exist either as a single nonassociating species or as a combination of assembly states independent of protein concentration. In the work described here, we examined the limit for ignoring weak reversible dimerization (Kd > or =1 mM) by comparing B values calculated with and without accounting for self-association. Light scattering effects for equilibrium dimer systems with Kd <20 mM and Kd <1 mM will significantly affect apparent B values measured for 20 and 150-kDa proteins, respectively. To interpret correctly light scattering data for monomer-dimer equilibrium systems, we use an expanded coefficient model to account for separate monomer-monomer (B(22)), monomer-dimer (B(23)), and dimer-dimer (B(33)) interactions.     

Dimer formation by a "monomeric" protein

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 10(5)-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 degrees C results in a 10(3)-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 degrees C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer-dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 degrees C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of "monomeric" proteins could be more prevalent than is usually appreciated.     

Catalytically active monomer of class mu glutathione transferase from rat

Although rat glutathione transferase M1-1 is crystallized as a homodimer (GST M1-1), we have generated monomers (GST M1) of the enzyme by adding potassium bromide to buffer solutions containing the wild-type enzyme and by introducing point mutations in the electrostatic region of the subunit interface. The wild-type enzyme was evaluated in 0.05 M MES (pH 6.5) containing up to 3 M KBr. We report that the addition of KBr greatly influences the monomer-dimer equilibrium of the wild-type enzyme and that at 3 M KBr GST M1 has a specific activity close to that of GST M1-1. Since the effect of KBr is likely due to charge screening at the subunit interface, the influence on the monomer-dimer equilibrium exerted by the amino acid residues in the electrostatic region of the interface (Arg77, Asp97, Glu100, Asn101) was investigated. Mutations introduced at positions 97, 100, and 101 promote monomerization, resulting in enzymes that exhibit a decreased weight average molecular weight in comparison to that of the wild-type enzyme. However, only mutations at position 97 result in enzymes that have catalytic activity in the monomeric form. The mutations introduced at positions 100 or 101 result in enzymes whose activity can be accounted for by the amount of dimeric enzyme present. Our results indicate that the electrostatic region of the interface is important in the monomer-dimer equilibrium of glutathione transferase and that, although GST M1-1 may be more active than GST M1, the dimer is not required for catalytic function.     

Slow association-dissociation equilibrium of NADP-linked isocitrate dehydrogenase from beef liver in relation to catalytic activity.

In this paper experiments are reported which show evidence for a relation between quaternary structure and catalytic activity of cytoplasmic NADP-linked isocitrate dehydrogenase from beef liver. The inactivation of the enzyme occurring upon dilution and the plots of the catalytic activity versus the enzyme concentration indicate that the monomeric species is catalytically inactive and that the monomer-dimer equilibrium is shifted towards the dimer upon binding of the substrate magnesium isocitrate complex. The association of the enzyme following binding of the substrate takes place at a rate comparable with that of the enzymatic reaction, which results in a 'hysteretic' behaviour of the enzyme. The possibility is discussed that slow changes in quaternary structure can give rise to a physiological regulation of the enzymatic activity.