A new family of cyanobacterial penicillin-binding proteins. A missing link in the evolution of class A beta-lactamases

It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and beta-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a beta-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A beta-lactamases. Interestingly, the Omega-loop, which, in the beta-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate beta-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter Omega-loop.     

Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates

Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin-activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha-lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha-lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added.     

Catalysis by serine proteases and their zymogens. A study of acyl intermediates by circular dichroism.

p-Nitrophenyl p'-guanidinobenzoate and methylumbelliferyl p'-guanidinobenzoate, which are active site titrants for trypsin, and p-nitrophenyl p'-dimethylsulfonioacetamidobenzoate and methylumbelliferyl p'-trimethylammoniocinnamate, which are active site titrants for chymotrypsin, are also hydrolyzed by the respective zymogens. Hydrolysis in each case proceeds via the formation of acyl-zymogens. The acylation rates for the zymogens are 10(3)-10(7) times slower than for the enzymes whereas the deacylation rates of acyl-enzymes and acyl-zymogens are comparable. These findings are consistent with the idea that the diminished catalytic activity of these zymogens is due primarily to their distorted substrate binding sites. The circular dichroic spectra of the acyl-enzymes show induced negative ellipticities in the region of absorption of the acyl group, due to binding of the group in an asymmetric environment. The circular dichroic spectra of the acyl-zymogens do not, but conversion of the acyl-zymogens to acyl-enzymes changes the circular dichroic spectra to those characteristic of the acyl-enzymes. alpha-Carbamyl-epsilon-guanidinated trypsin is a derivative which resembles trypsinogen in lacking activity toward specific ester substrates but possessing low activity toward p-nitrophenyl p'-guanidinobenzoate. The circular dichroic spectrum of the acyl-enzyme formed during hydrolysis of p-nitrophenyl p'-guanidinobenzoate by this derivative resembles that of guanidinobenzoyltrypsinogen, and not that of guanidinobenzoyltrypsin. These circular dichroism studies confirm that the same serine residue is involved in catalysis by both enzymes and zymogens. They demonstrate directly that the acylating group is in a different environment in each and indicate that this specific environment is a determinant in the catalytic activity of each. Thus the circular dichroic spectra of these acyl intermediates provide a sensitive probe of the subtle conformational changes which occur on zymogen activation. The results support the previous conclusion that the major feature of the activation of trypsinogen and chymotrypsinogen is the rearrangement of the substrate binding site and that the appearance of a new amino terminus causes this rearrangement.     

Direct observation of the titration of substrate carbonyl groups in the active site of alpha-chymotrypsin by resonance Raman spectroscopy

By use of resonance Raman (RR) spectroscopy, the population of the reactive carbonyl group in active acylchymotrypsins has been characterized and correlated with acyl-enzyme reactivity. RR spectra have been obtained, with a flow system and 324- and 337.5-nm excitation, at low and active pH for six acylchymotrypsins, viz., (indoleacryloyl)-, (4-amino-3-nitrocinnamoyl)-, (furylacryloyl)-, [( 5-ethylfuryl)-acryloyl]-, (thienylacryloyl)-, and [( 5-methylthienyl)acryloyl]chymotrypsin. These acyl-enzymes represent a 100-fold range of deacylation rate constants. Good RR spectral quality has enabled us to obtain the vibrational spectrum of the carbonyl group at low and active pH in each acyl-enzyme. The measured pKa of the spectroscopic changes in the carbonyl region is identical with that for the deacylation kinetics, showing that the RR carbonyl features reflect the ionization state of His-57. A carbonyl population has been observed in the active acyl-enzymes in which the carbonyl oxygen atom of the reactive acyl linkage is hydrogen-bonded in the active site. The proportion of this hydrogen-bonded population, with respect to other observed non-hydrogen-bonded species, together with the degree of polarization of the carbonyl bond, as monitored by vC = 0, has been correlated with the deacylation rate constants of the acyl-enzymes. It is proposed that the hydrogen-bonded carbonyl species is located at or near the oxyanion hole and represents the ground state from which deacylation occurs. An increase in the proportion of the hydrogen-bonded population and an increase in polarization of the carbonyl bond result in an increase in deacylation rate constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus

A keratinolytic protease from the fungus Doratomyces microsporus was investigated for its ability to hydrolyse different native proteins. The purified enzyme was incubated for up to 24 h with keratinous substrates as well as with non-keratinous proteins. The results showed that the enzyme was broad specific since it hydrolysed various globular and fibrillar proteins. The hydrolysis of keratinous substrates decreased in the following order: skin keratins > nail keratins > hair keratins. With non-keratinous substrates, the order was: casein > BSA > elastin. Feather keratin and collagen could not be hydrolysed. Comparison of the enzyme with some known proteolytic enzymes showed that on keratin from stratum corneum the activity of the keratinase was comparable to that of proteinase K, other enzymes were less active. Hydrolysis of porcine skin with the keratinase revealed the degradation of the epidermis while dermis was not damaged.     

Specificity of two isolated wheat carboxypeptidases

The substrate specificity of two purified carboxypeptidases from germinated wheat has been examined. Both enzymes were active on a wide variety of carbobenzoxy substituted peptides but inactive with unsubstituted dipeptides. Neither enzyme was active upon endoprotease or amidase substrates and only low levels of esterase activity were evident. In time course studies, both enzymes gave rapid non-specific sequential release of amino acids, including proline, from the carboxyterminal of proteins and polypeptides of known amino acid sequence.     

Hydrogen-bonding in enzyme catalysis. Fourier-transform infrared detection of ground-state electronic strain in acyl-chymotrypsins and analysis of the kinetic consequences.

I.r. difference spectra are presented for 3-(indol-3-yl)acryloyl-, cinnamoyl-, 3-(5-methylthien-2-yl)acryloyl-, dehydrocinnamoyl- and dihydrocinnamoyl-chymotrypsins at low pH, where the acyl-enzymes are catalytically inactive. At least two absorption bands are seen in each case in the ester carbonyl stretching region of the spectrum. Cinnamoyl-chymotrypsin substituted at the carbonyl carbon atom with 13C was prepared. A difference spectrum in which 13C-substituted acyl-enzyme was subtracted from [12C]acyl-enzyme shows two bands in the ester carbonyl region and thus confirms the assignment of the features to the single ester carbonyl group. The frequencies of the ester carbonyl bands are interpreted in terms of differential hydrogen-bonding. In each case a lower-frequency relatively narrow band is assigned to a productive potentially reactive binding mode in which the carbonyl oxygen atom is inserted in the oxyanion hole of the enzyme active centre. The higher-frequency band, which is broader, is assigned to a non-productive binding mode in each case, where a water molecule bridges from the carbonyl oxygen atom to His-57; this mode is equivalent to the crystallographically determined structure of 3-(indol-3-yl)acryloyl-chymotrypsin, i.e. the Henderson structure. A difference spectrum of dihydrocinnamoyl-chymotrypsin taken at higher pH shows resolution of a feature centred upon 1731 cm-1, which is assigned to a non-bonded conformer in which the carbonyl oxygen atom is not hydrogen-bonded. Perturbation of the protein spectrum in the presence of acyl groups is interpreted in terms of enhanced structural rigidity. It is reported that the ester carbonyl region of the difference spectrum of cinnamoyl-subtilisin is complicated by overlap of features that arise from protein perturbation. Measurements of carbonyl absorption frequencies in a number of solvents of the methyl esters of the acyl groups used to make acyl-enzymes have permitted determination of the apparent dielectric constants experienced by carbonyl groups in the enzyme active centre as well as a discussion of the effects of polarity. The ester carbonyl bond strengths of the various conformations were estimated by using simple harmonic oscillator theory and an empirical relation between the force constants and bond strengths. The fractional bond breaking induced by hydrogen-bonding was used to calculate rate enhancement factors by using absolute reaction rate theory.(ABSTRACT TRUNCATED AT 400 WORDS)     

A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains.

Molecular modeling based on the crystal structure of the Rous sarcoma virus (RSV) protease dimer has been used to link the two identical subunits of this enzyme into a functional, single polypeptide chain resembling the nonviral aspartic proteases. Six different linkages were selected to test the importance of different interactions between the amino acids at the amino and carboxyl termini of the two subunits. These linkages were introduced into molecular clones of fused protease genes and the linked protease dimers were expressed in Escherichia coli and purified. Catalytically active proteins were obtained from the inclusion body fraction after renaturation. The linked protease dimers exhibited a 10-20-fold range in catalytic efficiencies (Vmax/Km) on peptide substrates. Both flexibility and ionic interactions in the linkage region affect catalytic efficiency. Some of the linked protease dimers were 2-3-fold more active than the nonlinked enzyme purified from bacteria, although substrate specificities were unchanged. Similar relative efficiencies were observed using a polyprotein precursor as substrate. Mutation of one catalytic Asp in the most active linked protease dimer inactivated the enzyme, demonstrating that these proteins function as single polypeptide chains rather than as multimers.     

Length of the acyl carbonyl bond in acyl-serine proteases correlates with reactivity

Resonance Raman (RR) spectroscopy has been used to obtain the vibrational spectrum of the acyl carbonyl group in a series of acylchymotrypsins and acylsubtilisins at the pH of optimum hydrolysis. The acyl-enzymes, which utilize arylacryloyl acyl groups, include three oxyanion hole mutants of subtilisin BPN', Asn155Leu, Asn155Gln, and Asn155Arg, and encompass a 500-fold range of deacylation rate constants. For each acyl-enzyme a RR carbonyl band has been identified which arises from a population of carbonyl groups undergoing nucleophilic attack in the active site. As the deacylation rate (k3) increases through the series of acyl-enzymes, the carbonyl stretching band (vC = O) is observed to shift to lower frequency, indicating an increase in single bond character of the reactive acyl carbonyl group. Experiments involving the oxyanion hole mutants of subtilisin BPN' indicate that a shift of vC = O to lower frequency results from stronger hydrogen bonding of the acyl carbonyl group in the oxyanion hole. A plot of log k3 against vC = O is linear over the range investigated, demonstrating that the changes in vC = O correlate with the free energy of activation for the deacylation reaction. By use of an empirical correlation between carbonyl frequency (vC = O) and carbonyl bond length (rC = O) it is estimated that rC = O increases by 0.015 A as the deacylation rate increases 500-fold through the series of acyl-enzymes. This change in rC = O is about 7% of that expected for going from a formal C = O double bond in the acyl-enzyme to a formal C-O single bond in the tetrahedral intermediate for deacylation. The data also allow us to estimate the energy needed to extend the acyl carbonyl group along its axis to be 950 kJ mol-1 A-1.     

Human endothelial cells contain one type of plasminogen activator

RNA-binding protein kinase from amphibian oocytes modifies serine and threonine residues in the molecules of substrates and utilizes both ATP and GTP. Low concentrations of heparin inhibit protein kinase. The foregoing suggests that this enzyme is casein kinase II. It is shown that RNA-binding proteins lack active forms of phosphatases and proteases which may affect the results of phosphorylation of both endogenous and exogenous substrates.     

Inhibition of beta-lactamase by clavulanate. Trapped intermediates in cryocrystallographic studies.

Crystallographic studies of the complex between beta-lactamase and clavulanate reveal a structure of two acyl-enzymes with covalent bonds at the active site Ser70, representing two different stages of inhibitor degradation alternately occupying the active site. Models that are consistent with biochemical data are derived from the electron density map and refined at 2.2 A resolution: cis enamine, in which the carboxylate group of the clavulanate molecule makes a salt bridge with Lys234 of beta-lactamase; decarboxylated trans enamine, which is oriented away from Lys234. For both acyl-enzymes, the carbonyl oxygen atom of the ester group occupies the oxyanion hole in a manner similar to that found in inhibitor binding to serine proteases. Whereas the oxygen atom in the trans product is optimally positioned in the oxyanion hole, that of the cis product clashes with the main-chain nitrogen atom of Ser70 and the beta-carbon atom of the adjacent Ala69. In contrast to cis to trans isomerization in solution that relieves the steric strain inherent in a cis double bond, at the enzyme-inhibitor interface two additional factors play an important role. The salt bridge enhances the stability of the cis product, while the steric strain introduced by the short contacts with the protein reduces its stability.     

A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues

The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins.     

The acyl-CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p

One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay. These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized.     

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).     

Streptococcus pneumoniae DNA polymerase I lacks 3''-to-5'' exonuclease activity: localization of the 5''-to-3'' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.     

Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop

A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue.     

Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1(-/-) mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-(3)H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1(-/-) animals compared with Pcmt1(+/+) littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, beta-synuclein, and alpha-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1(-/-) brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.     

Inactivation and stabilization of stabilisins in neat organic solvents

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.     

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [γ³²P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.     

Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein.

Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(U)--agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)--AGAROSe. Moreover, the native enzyme linked on blue-dextran--Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran--Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.