A kinetic analysis of the oligonucleotide-modulated ATPase activity of the helicase domain of the NS3 protein from hepatitis C virus. The first cycle of interaction of ATP with the enzyme is unique

Hepatitis C virus (HCV) helicase (E) formed spectrofluorometrically detectable complexes with a 16-mer and HF16 (a 16-mer with 5'-hexachlorofluoresceinyl moiety). The interaction of helicase with these effectors was investigated by kinetic techniques to determine if the complexes were kinetically competent for ATP hydrolysis. kcat values with the 16-mer and HF16 were 2.7 and 36 s-1, respectively. The maximal value of the rate constant for the approach of an intermediate to the steady-state level has to be at least 4-fold greater than kcat for it to be kinetically competent. This value was 1.2 s-1 with HF16 and "E.ATP" and was 1.82 s-1 with ATP and E.HF16. These values were too small for formation of these intermediates to be kinetically competent in ATP hydrolysis. Dissociation of "E.HF16. ATP" (0.34 s-1) was also too slow to contribute significantly to catalysis. Furthermore, the Km of E.HF16 for ATP (3 mircoM) was significantly less than the Km for ATP hydrolysis at a saturating concentration of HF16 (320 microM). HCV helicase has two nucleotide-binding sites per monomer. If the fluorescence changes observed were associated with structure changes preceding steady-state catalysis (isomerization), pre-steady-state data could be reconciled with the turnover data. Data for the 16-mer yielded similar conclusions.     

Inhibitory mechanism of serpins: loop insertion forces acylation of plasminogen activator by plasminogen activator inhibitor-1

Serpin inhibitors are believed to form an acyl enzyme intermediate with their target proteinases which is stabilized through insertion of the enzyme-linked part of the reactive center loop (RCL) as strand 4 in beta-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in beta-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for both tissue-type plasminogen activator (tPA) and trypsin and was hydrolyzed at the scissile bond. The reactivity of the peptide-blocked substrate PAI-1 was compared to that of the unmodified inhibitor by rapid acid quenching as well as photometric techniques. With trypsin as target, the limiting rate constants for enzyme acylation were essentially the same with inhibitor and substrate PAI-1 (21-23 s-1), as were also the associated apparent second-order rate constants (2.8-2.9 microM-1 s-1). With tPA, inhibitor and substrate PAI-1 reacted identically to form a tightly bound Michaelis complex (Kd approximately Km approximately 20 nM). The limiting rate constant for acylation of tPA, however, was 57 times faster with inhibitor PAI-1 (3.3 s-1) than with the substrate form (0.059 s-1), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 microM-1 s-1). We attribute the ability of tPA to discriminate between the two PAI-1 forms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of tPA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bonds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.     

Simultaneous formation of functional leading and lagging strand holoenzyme complexes on a small, defined DNA substrate

The biochemical characterization of leading and lagging strand DNA synthesis by bacteriophage T4 replication proteins has been addressed utilizing a small, defined primer/template. The ATP hydrolysis activity of 44/62, the clamp loading complex responsible for holoenzyme assembly, was monitored during assembly of both the leading and lagging strand holoenzyme complex. The ATPase activity of 44/62 diminishes once a functional holoenzyme is assembled on both the leading and lagging strand. The assembly of the lagging strand holoenzyme is facilitated by several factors including biotinylated streptavidin blocks at the end of the fork strands, preassembly of the leading strand holoenzyme, and by the presence of the DNA primase with ribonucleoside triphosphates. The resultant minimal replicative complex consists of two holoenzymes and a primase nested on a model replication fork derived from a 62-mer template/34-mer primer/36-mer lagging strand in an apparent 2:2:1:1 ratio of 45 protein:polymerase:primase:forked DNA. The 44/62 protein complex does not remain associated with the complex. The primase alone slowly synthesizes pentaribonucleotides on the forked DNA when the lagging strand contains a nonannealed TTG initiation site with the rate of synthesis greatly stimulated by the addition of the 41 helicase. The addition of deoxy-NTPs to this complex results in leading strand synthesis, but extension of the synthesized RNA primer does not occur. DNA synthesis in both the leading and lagging strand directions is achieved, however, when a 6-mer DNA primer is annealed to the primase recognition site of the forked DNA substrate. A model is presented that describes how leading and lagging strand DNA synthesis might be coordinated as well as the associated molecular interactions of the replicative proteins.     

Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains

Sam68, a specific target of the Src tyrosine kinase in mitosis, possesses features common to RNA-binding proteins, including a K homology (KH) domain. To elucidate its biological function, we first set out to identify RNA species that bound to Sam68 with high affinity using in vitro selection. From a degenerate 40-mer pool, 15 RNA sequences were selected that bound to Sam68 with Kd values of 12-140 nM. The highest affinity RNA sequences (Kd approximately 12-40 nM) contained a UAAA motif; mutation to UACA abolished binding to Sam68. Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding. The KH domain alone did not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding. Deletion of the KH domain or mutation of KH domain residues analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-1 abolished G8-5 binding. Our results establish that a KH domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development.     

Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain contains the low affinity site. Also, binding of TIMP-2 to pro-MMP-2 is stronger at both the high and low affinity sites than the corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM at the high affinity site, yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP-1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics. This suggests that a single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.     

Dissociation of bacteriophage T4 DNA polymerase and its processivity clamp after completion of Okazaki fragment synthesis

The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki fragment. Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5'-end primer contained either a 5'-OH (DNA primer) or a 5'-triphosphate (RNA primer) group. Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (approximately 40%) or strand-displaces the blocking strand (approximately 60%). The two blocking oligonucleotides (DNA or RNA) induce a 30-50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously. Inhibition of ATP hydrolysis by ATP-gamma-S did not have a measurable effect upon holoenzyme dissociation from DNA. The presence of gp32, the single-strand binding protein, caused a small (3-fold) increase in the rate constant for dissociation.     

Interaction of heparan sulfate from mammary cells with acidic fibroblast growth factor (FGF) and basic FGF. Regulation of the activity of basic FGF by high and low affinity binding sites in heparan sulfate

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.     

Tight binding of folate substrates and inhibitors to recombinant mouse glycinamide ribonucleotide formyltransferase

The binding of the prototypical folate inhibitor of de novo purine synthesis, 5,10-dideazatetrahydrofolate (DDATHF), and its hexaglutamate to recombinant trifunctional mouse glycinamide ribonucleotide formyltransferase (rmGARFT) was studied by equilibrium dialysis and by steady-state kinetics using sensitive assays that allowed initial rate calculations. rmGARFT was expressed in insect cells infected with a recombinant baculovirus and purified by a two-step procedure that allowed production of about 25 mg of pure protein/L of culture. The binding of DDATHF to GARFT was approximately 50-fold tighter than previously reported, with Kd and Ki values of 2-9 nM, making the parent form of this antifolate a tight-binding inhibitor. The binding of the hexaglutamate of DDATHF to rmGARFT had Kd and Ki values of 0.1-0.3 nM, consistent with the view that polyglutamation enhances binding of antifolates to GARFT. Kinetic analyses using either mono- or hexaglutamate substrate did not yield different values for the Ki for the hexaglutamate form of DDATHF, in contradiction with previous reports. Both the folate substrate commonly used to study GARFT, 10-formyl-5,8-dideazafolate, and its hexaglutamate were found to have very low Km values, namely, 75 and 7.4 nM, respectively, and the folate reaction products for these substrates were equally potent inhibitors, results which modify the interpretation of previous kinetic experiments. The product analog DDATHF and beta-glycinamide ribonucleotide bound to enzyme equally well in the presence and absence of the other, an observation at variance with the concept that GARFT obeys an ordered sequential binding of the substrates. We conclude that the kinetics of mouse GARFT are most consistent with a random order of substrate binding, that both the inhibitor DDATHF and the folate substrate are tight-binding ligands, and that polyglutamate forms enhance the affinity of both substrate and inhibitor by an order of magnitude.     

Hepatocyte growth factor/scatter factor has distinct classes of binding site in heparan sulfate from mammary cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan sulfate (HS)-binding growth factor and morphogen for mammary epithelial cells that is produced by mammary stromal fibroblasts. HS chains, purified as peptidoglycans from a panel of cell lines representative of the ductal epithelial cell (Huma 123), the myoepithelial cell (Huma 109), the stromal fibroblast (Rama 27), and malignant mammary epithelial cells (MCF-7 and ZR-75), were used in a biosensor-based assay to identify the classes of HGF/SF-binding sites in the polysaccharide chains. At least three distinct binding sites were identified. One site exhibits fast association and fast dissociation kinetics [kass (1.4-7.7) x 10(6) M-1 s-1; kdiss 0. 0032-0.0096 s-1] and is present on the HS from benign Huma 123 epithelial cells, Huma 109 myoepithelial-like cells, and ZR-75 malignant cells. The second binding site, found on HS from the malignant MCF-7 cells, has slower HGF/SF-binding kinetics (kass 0.20 x 10(6) M-1 s-1; kdiss 0.00055 s-1). The third binding site possesses fast association and slow dissociation kinetics (kass 1.1 x 10(6) M-1 s-1; kdiss 0.00020 s-1) and was found on the HS isolated from the culture medium of the Huma 123 benign epithelial cells. The first and second binding sites have a similar Kd, 1-3 nM, while the third binding site has a considerably higher affinity for HGF/SF (Kd 200 pM). The three binding sites seem to be mutually exclusive, since each sample of HS possessed just one of the sites.     

Telomerase protein rather than its RNA is the target of phosphorothioate-modified oligonucleotides

Human telomerase is a ribonucleoprotein which uses its internal RNA moiety as a template for telomeric DNA synthesis. This enzyme is up-regulated in most malignant tumors and is therefore considered as a possible cancer target. Here we examined the effects of differently modified oligomers on telomeraseactivity from HL-60 cell extracts (TRAP-ezetrade mark assay). Phosphorothioate-modified oligonucleotides (PS-ODNs) inhibited telomerase activity at subnanomolar concen-trations and proved to be more efficient than peptide nucleic acids. In contrast to all the investigated oligomers, PS-ODNs were found to bind to the protein motif of telomerase called the primer binding site but poorly to its RNA. This is suggested by kinetic investigations demonstrating a competitive interaction of PS-ODNs and TS primer at the primer binding site. The K m value of the TS primer was 10.8 nM, the K i value of a 20mer PS-ODN was 1.6 nM. When the TS primer was PS-modified a striking increase in the telomerase activity was found which correlates with the number of phosphodiesters replaced. The K m value of a completely PS-modified TS primer was 0.56 nM. Based on these results the design of chimeric ODNs is proposed consisting of a 5'-PS-modified part targeting the primer binding site and a 3'-terminus part targeting the telomerase RNA.     

Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.     

Intrastriatal adenosine A1 receptor antisense oligodeoxynucleotide blocks ethanol-induced motor incoordination

Intrastriatal administration of a 21-mer phosphorothioate antisense oligodeoxynucleotide targeting the adenosine A1 receptor blocked ethanol-induced motor incoordination in the rat and reduced striatal adenosine A1 receptor content, as judged by specific binding of the A1-specific ligand 8-cyclopentyl-1,3-dipropylxanthine (Bmax = 0.350 +/- 0.07, Kd = 1.87 +/- 0.50 nM). No effect upon striatal adenosine A2 receptor content was observed (Bmax = 0.415 +/- 0.04, Kd = 13.13 +/- 1.25 nM) with the A2-specific ligand 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine. A mismatched control oligodeoxynucleotide of identical G-C base composition and general sequence structure was without effect on adenosine A1 receptor (Bmax = 0.666 +/- 0.11, Kd = 1.32 +/- 0.27 nM) or adenosine A2 receptor content (Bmax = 0.501 +/- 0.08; Kd = 14.65 +/- 1.82 nM) or ethanol-induced motor incoordination. These results confirm an important role of the striatal adenosine A1 receptor in mediating certain motor-related physiological effects of ethanol.