Interaction of the adenovirus proteinase with protein cofactors with high negative charge densities

The interactions of the human adenovirus proteinase (AVP) with polymers with high negative charge densities were characterized. AVP utilizes two viral cofactors for maximal enzyme activity (k(cat)/K(m)), the 11-amino acid peptide from the C-terminus of virion precursor protein pVI (pVIc) and the viral DNA. The viral DNA stimulates covalent AVP-pVIc complexes (AVP-pVIc) as a polyanion with a high negative charge density. Here, the interactions of AVP-pVIc with different polymers with high negative charge densities, polymers of glutamic acid (polyE), were characterized. The rate of substrate hydrolysis by AVP-pVIc increased with increasing concentrations of polyE. At higher concentrations of polyE, the increase in the rate of substrate hydrolysis approached saturation. Although glutamic acid did not stimulate enzyme activity, glutamic acid and NaCl could displace DNA from AVP-pVIc.(DNA) complexes; the K(i) values were 230 and 329 nM, respectively. PolyE binds to the DNA binding site on AVP-pVIc as polyE and DNA compete for binding to AVP-pVIc. The equilibrium dissociation constant for 1.3 kDa polyE binding to AVP-pVIc was 56 nM. On average, one molecule of AVP-pVIc binds to 12 residues in polyE. Comparison of polyE and 12-mer single-stranded DNA interacting with AVP-pVIc revealed the binding constants are similar, as are the Michaelis-Menten constants for substrate hydrolysis. The number of ion pairs formed upon the binding of 1.3 kDa polyE to AVP-pVIc was 2, and the nonelectrostatic change in free energy upon binding was -6.5 kcal. These observations may be physiologically relevant as they infer that AVP may bind to proteins that have regions of negative charge density. This would restrict activation of the enzyme to the locus of the cofactor within the cell.     

Interaction of the human adenovirus proteinase with its 11-amino acid cofactor pVIc

The interaction of the human adenovirus proteinase (AVP) and AVP-DNA complexes with the 11-amino acid cofactor pVIc was characterized. The equilibrium dissociation constant for the binding of pVIc to AVP was 4.4 microM. The binding of AVP to 12-mer single-stranded DNA decreased the K(d) for the binding of pVIc to AVP to 0.09 microM. The pVIc-AVP complex hydrolyzed the substrate with a Michaelis constant (K(m)) of 3.7 microM and a catalytic rate constant (k(cat)) of 1.1 s(-1). In the presence of DNA, the K(m) increased less than 2-fold, and the k(cat) increased 3-fold. Alanine-scanning mutagenesis was performed to determine the contribution of individual pVIc side chains in the binding and stimulation of AVP. Two amino acid residues, Gly1' and Phe11', were the major determinants in the binding of pVIc to AVP, while Val2' and Phe11' were the major determinants in stimulating enzyme activity. Binding of AVP to DNA greatly suppressed the effects of the alanine substitutions on the binding of mutant pVIcs to AVP. Binding of either or both of the cofactors, pVIc or the viral DNA, to AVP did not dramatically alter its secondary structure as determined by vacuum ultraviolet circular dichroism. pVIc, when added to Hep-2 cells infected with adenovirus serotype 5, inhibited the synthesis of infectious virus, presumably by prematurely activating the proteinase so that it cleaved virion precursor proteins before virion assembly, thereby aborting the infection.     

DNA binding provides a molecular strap activating the adenovirus proteinase

Human adenovirus proteinase (AVP) requires two cofactors for maximal activity: pVIc, a peptide derived from the C terminus of adenovirus precursor protein pVI, and the viral DNA. Synchrotron protein footprinting was used to map the solvent accessible cofactor binding sites and to identify conformational changes associated with the binding of cofactors to AVP. The binding of pVIc alone or pVIc and DNA together to AVP triggered significant conformational changes adjacent to the active site cleft sandwiched between the two AVP subdomains. In addition, upon binding of DNA to AVP, it was observed that specific residues on each of the two major subdomains were significantly protected from hydroxyl radicals. Based on the locations of these protected side-chain residues and conserved aromatic and positively charged residues within AVP, a three-dimensional model of DNA binding was constructed. The model indicated that DNA binding can alter the relative orientation of the two AVP domains leading to the partial activation of AVP by DNA. In addition, both pVIc and DNA may independently alter the active site conformation as well as drive it cooperatively to fully activate AVP.     

Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.

The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc.     

Interaction of actin and its 11-amino acid C-terminal peptide as cofactors with the adenovirus proteinase

Actin bound to the adenovirus proteinase (AVP) with a lower equilibrium dissociation constant, 4.2 nM, than those exhibited by two viral, nuclear cofactors for AVP, the 11-amino acid peptide pVIc and the viral DNA. The k(cat)/K(m) ratio for substrate hydrolysis by AVP increased 150,000-fold in the presence of actin. The 11-amino acid residue peptide corresponding to the C-terminus of actin, which is highly homologous to pVIc, bound to AVP and stimulated its activity in the presence of DNA. As a cellular cofactor for AVP, AVP(actin) complexes may facilitate the cleavage of cytoskeletal proteins, preparing the infected cell for lysis and release of nascent virions.     

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space: III. ATOMIC RESOLUTION STRUCTURE OF THE NASCENT FORM OF THE ADENOVIRUS PROTEINASE*

The adenovirus proteinase (AVP), the first member of a new class of cysteine proteinases, is essential for the production of infectious virus, and here we report its structure at 0.98 Å resolution. AVP, initially synthesized as an inactive enzyme, requires two cofactors for maximal activity: pVIc, an 11-amino acid peptide, and the viral DNA. Comparison of the structure of AVP with that of an active form, the AVP-pVIc complex, reveals why AVP is inactive. Both forms have an α + β fold; the major structural differences between them lie in the β-sheet domain. In AVP-pVIc, the general base His-54 Nδ1 is 3.9 Å away from the Cys-122 Sγ, thereby rendering it nucleophilic. In AVP, however, His-54 Nδ1 is 7.0 Å away from Cys-122 Sγ, too far away to be able to abstract the proton from Cys-122. In AVP-pVIc, Tyr-84 forms a cation-π interaction with His-54 that should raise the pK_a of His-54 and freeze the imidazole ring in the place optimal for forming an ion pair with Cys-122. In AVP, however, Tyr-84 is more than 11 Å away from its position in AVP-pVIc. Based on the structural differences between AVP and AVP-pVIc, we present a model that postulates that activation of AVP by pVIc occurs via a 62-amino acid-long activation pathway in which the binding of pVIc initiates contiguous conformational changes, analogous to falling dominos. There is a common pathway that branches into a pathway that leads to the repositioning of His-54 and another pathway that leads to the repositioning of Tyr-84.     

Crystallographic structure at 1.6-A resolution of the human adenovirus proteinase in a covalent complex with its 11-amino-acid peptide cofactor: insights on a new fold

The crystal structure of the human adenovirus proteinase (AVP), a cysteine proteinase covalently bound to its 11-amino-acid peptide cofactor pVIc, has been solved to 1.6-A resolution with a crystallographic R-factor of 0.136, R(free)=0.179. The fold of AVP-pVIc is new and the structural basis for it is described in detail. The polypeptide chain of AVP folds into two domains. One domain contains a five-strand beta-sheet with two peripheral alpha-helices; this region represents the hydrophobic core of the protein. A second domain contains the N terminus, several C-terminal alpha-helices, and a small peripheral anti-parallel beta-sheet. The domains interact through an extended polar interface. pVIc spans the two domains like a strap, its C-terminal portion forming a sixth strand on the beta-sheet. The active site is in a long, deep groove located between the two domains. Portions are structurally similar to the active site of the prototypical cysteine proteinase papain, especially some of the Calpha backbone atoms (r.m.s. deviation of 0.354 A for 12 Calpha atoms). The active-site nucleophile of AVP, the conserved Cys(122), was shown to have a pK(a) of 4.5, close to the pK(a) of 3.0 for the nucleophile of papain, suggesting that a similar ion pair arrangement with His(54) may be present in AVP-pVIc. The interactions between AVP and pVIc include 24 non-beta-strand hydrogen bonds, six beta-strand hydrogen bonds and one covalent bond. Of the 204 amino acid residues in AVP, 33 are conserved among the many serotypes of adenovirus, and these aid in forming the active site groove, are involved in substrate specificity or interact between secondary structure elements.     

Crystal structure of the human adenovirus proteinase with its 11 amino acid cofactor.

The three-dimensional structure of the human adenovirus-2 proteinase complexed with its 11 amino acid cofactor, pVIc, was determined at 2.6 A resolution by X-ray crystallographic analysis. The fold of this protein has not been seen before. However, it represents an example of either subtly divergent or powerfully convergent evolution, because the active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. Thus, the adenovirus proteinase represents a new, fifth group of enzymes that contain catalytic triads. pVIc, which extends a beta-sheet in the main chain, is distant from the active site, yet its binding increases the catalytic rate constant 300-fold for substrate hydrolysis. The structure reveals several potential targets for antiviral therapy.     

Hepatitis A virus 3C proteinase substrate specificity.

Hepatitis A virus (HAV) 3C proteinase is responsible for processing the viral precursor polyprotein into mature proteins. The substrate specificity of recombinant hepatitis A 3C proteinase was investigated using a series of synthetic peptides representing putative polyprotein junction sequences. Two peptides, corresponding to the viral polyprotein 2B/2C and 2C/3A junctions, were determined to be cleaved most efficiently by the viral 3C proteinase. The kcat/Km values determined for the hydrolysis of a further series of 2B/2C peptides, in which C-terminal and N-terminal amino acids were systematically removed, revealed that P4 through P2' amino acids were necessary for efficient substrate cleavage. The substitution of Ala for amino acids in P1 and P4 positions decreased the rate of peptide hydrolysis by 100- and 10-fold, respectively, indicating that the side chains of Gln in P1 and Leu in P4 are important determinants of substrate specificity. Rates of hydrolysis measured for other P1- and P4-substituted peptides indicate that S1 is very specific for the Gln side chain whereas S4 requires only that the amino acid in P4 be hydrophobic. A continuous fluorescence quench assay was developed, allowing the determination of kcat/Km dependence on pH. The pH rate profile suggests that catalyzed peptide hydrolysis is dependent on deprotonation of a reactive group having a pKa of 6.2 (+/- 0.2). The results of tests with several proteinase inhibitors indicate that this cysteine proteinase, like other picornaviral 3C proteinases, is not a member of the papain family.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

Deubiquitinating function of adenovirus proteinase

The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Specificity studies on retroviral proteinase from myeloblastosis-associated virus.

The specificity of the p15 proteinase of myeloblastosis-associated virus (MAV) was tested with nonviral high molecular weight substrates and with synthetic peptides. Peptides with sequences spanning known cleavage sites in viral polyproteins of Rous sarcoma virus (RSV) and avian leukemia viruses, as well as in BSA and HSA, were synthesized, and the rate of their cleavage by the MAV proteinase was compared. Synthetic peptides require for successful cleavage at least 4 residues at the N-terminal side and 3 residues at the C-terminal side. The proteinase shows a preference for hydrophobic residues with bulky side chains (Met, Tyr, Phe) in P3, although Arg and Gln can also be accepted. Small hydrophobic residues are required in P2 and P2', and large hydrophobic residues (Tyr, Met, Phe/p-nitro-Phe) are preferred in both P1 and P1'. The difference between the specificity of the p15 proteinase and that of the HIV-1 proteinase mostly pertains to position P2' of the substrate, where bulkier side chains are accepted by the HIV-1 proteinase (Richards et al., 1990). A good chromogenic substrate for the MAV and RSV proteinases was developed and used to further characterize the MAV proteinase activity with respect to ionic strength and pH. The activity of the proteinase is strongly dependent on ionic strength and pH. Both the kcat and Km values contribute to a higher cleavage efficiency at higher salt concentrations and show a bell-shaped pH dependence curve with a sharp maximum at pH 5.5 (kcat) and 6.5 (Km).     

Actin can act as a cofactor for a viral proteinase in the cleavage of the cytoskeleton

Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of the cytokeratin network. For AVP to cleave cytokeratin 18, a cellular cofactor was shown to be required, consistent with AVP being synthesized as an inactive proteinase. Actin was considered a cellular cofactor for AVP, because the C terminus of actin is homologous to a viral cofactor for AVP. AVP was shown to bind to the C terminus of actin, and in doing so AVP exhibited full enzymatic activity. In vitro, actin was a cofactor in the cleavage of cytokeratin 18 by AVP. The proteinase alone could not cleave cytokeratin 18, but in the presence of actin, AVP cleaved cytokeratin 18. Indeed, actin itself was shown to be a cofactor and a substrate for its own destruction in that it was cleaved by AVP in vitro. Cleavage of cytoskeletal proteins weakens the structure of the cell, and therefore, actin as a cofactor may play a role in cell lysis and release of nascent virions.     

Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.     

The Cleaved N-Terminus of pVI Binds Peripentonal Hexons in Mature Adenovirus

Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen–deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.     

Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp

The gene encoding a peptidase that belongs to the proteinase K family of serine peptidases has been identified from a psychrotrophic Serratia sp., and cloned and expressed in Escherichia coli. The gene has 1890 base pairs and encodes a precursor protein of 629 amino acids with a theoretical molecular mass of 65.5 kDa. Sequence analysis suggests that the peptidase consists of a prepro region, a catalytic domain and two C-terminal domains. The enzyme is recombinantly expressed as an active approximately 56 kDa peptidase and includes both C-terminal domains. Purified enzyme is converted to the approximately 34 kDa form by autolytic cleavage when incubated at 50 degrees C for 30 min, but retains full activity. In the present work, the Serratia peptidase (SPRK) is compared with the family representative proteinase K (PRK) from Tritirachium album Limber. Both enzymes show a relatively high thermal stability and a broad pH stability profile. SPRK possess superior stability towards SDS at 50 degrees C compared to PRK. On the other hand, SPRK is considerably more labile to removal of calcium ions. The activity profiles against temperature and pH differ for the two enzymes. SPRK shows both a broader pH optimum as well as a higher temperature optimum than PRK. Analysis of the catalytic properties of SPRK and PRK using the synthetic peptide succinyl-Ala-Ala-Pro-Phe-pNA as substrate showed that SPRK possesses a 3.5-4.5-fold higher kcat at the temperature range 12-37 degrees C, but a fivefold higher Km results in a slightly lower catalytic efficiency (kcat/Km) of SPRK compared to PRK.     

Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA

We find that the rate of dsDNA-dependent ATPase activity is biphasic, with a fast component which represents the unwinding of the dsDNA and a slow component which results from the ssDNA-dependent ATPase activity of recBCD enzyme. Comparison of the ATPase and helicase activities permits evaluation of the efficiency of ATP hydrolysis during unwinding. This efficiency can be calculated from the maximum rates of ATPase and helicase activities and is found to range between 2.0 and 3.0 ATP molecules hydrolyzed per base pair of DNA unwound. The number of ATP molecules hydrolyzed per base pair unwound is not altered by temperature but does increase at low concentrations of DNA and high concentrations of sodium chloride and magnesium acetate. The apparent Km values for the DNA and ATP substrates of recBCD enzyme dsDNA-dependent ATPase activity at 25 degrees C were determined to be 0.13 nM DNA molecules and 85 microM ATP, respectively. The observed kcat value is approximately 45 microM ATP s-1 (microM recBCD enzyme)-1. If this rate is corrected for the measured stoichiometry of recBCD enzyme binding to dsDNA, the kcat for ATPase activity corresponds to an ATP hydrolysis rate of approximately 740 ATP molecules s-1 (functional recBCD complex)-1 at 25 degrees C.     

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.