Differential translation of turnip yellow mosaic virus mRNAs in vitro

Total TYMV RNA was incubated in a reticulocyte lysate, and the initiation peptides of the main proteins synthesized $\text{in vitro}$ (195 K, 150 K and 20 K daltons) analyzed after tryptic digestion. The 195 K and the 150 K dalton proteins present analogous patterns, different from the one obtained with the 20 K dalton protein (coat protein), suggesting that only one initiation site exists on the genomic RNA for the synthesis of the two high molecular proteins. The results of competition experiments between genomic and coat protein mRNA indicate that the ribosomes have a much greater affinity for the coat protein mRNA. This may represent a regulatory mechanism for the preferential amplification of coat protein synthesis in the infected cells.     

The ADP-ribosylating mosquitocidal toxin from Bacillus sphaericus: proteolytic activation, enzyme activity, and cytotoxic effects.

The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is a approximately 97-kDa protein sharing sequence homology within the N terminus with the catalytic domains of various bacterial ADP-ribosyltransferases. Here we studied the proteolytic activation of the ADP-ribosyltransferase activity of MTX. Chymotrypsin treatment of the 97-kDa MTX holotoxin (MTX(30-870)) results in a 70-kDa putative binding component (MTX(265-870)) and a 27-kDa enzyme component (MTX(30-264)), possessing ADP-ribosyltransferase activity. Chymotryptic cleavage of an N-terminal 32-kDa fragment of MTX (MTX(30-308)) also yields MTX(30-264), but the resulting ADP-ribosyltransferase activity is much greater than that of the processed MTX(30-870). Kinetic studies revealed a K(m) NAD value of 45 microm for the processed 32-kDa MTX fragment, and a K(m) NAD value of 1300 microm for the processed holotoxin. Moreover, the k(cat) value for the activated MTX(30-308) fragment was about 10-fold higher than that for the activated holotoxin (MTX(30-870)). Precipitation analysis showed that the 70-kDa proteolytic fragment of MTX remains noncovalently bound to the N-terminal 27-kDa fragment, thereby inhibiting ADP-ribosyltransferase and NAD glycohydrolase activities. Glu(197) of MTX(30-264) was identified as the "catalytic" glutamate that is conserved in all ADP-ribosyltransferases. Whereas mutated MTX(30-264)E197Q has neither ADP-ribosyltransferase nor NAD glycohydrolase activity, mutated MTX(30-264)E195Q possesses glycohydrolase activity but not transferase activity. Transfection of HeLa cells with a vector encoding a fusion protein of MTX(30-264) with a green fluorescent protein led to cytotoxic effects characterized by cell rounding and formation of filopodia-like protrusions. These cytotoxic effects were not observed with the catalytically inactive MTX(30-264)E197Q mutant, indicating that the MTX enzyme activity is essential for the cytotoxicity in mammalian cells.     

Identification of a MHC class-II restricted epitope in carcinoembryonic antigen

Purpose The carcinoembryonic antigen (CEA) is extensively expressed on the vast majority of colorectal, gastric, and pancreatic carcinomas, and, therefore, is a good target for tumor immunotherapy. CD4+ T-helper (Th) cells play a critical role in initiation, regulation, and maintenance of immune responses. In this study, we sought to identify Th epitopes derived from CEA which can induce CEA-specific Th responses. The combined application with cytotoxic T lymphocyte (CTL) epitopes would be more potent than tumor vaccines that primarily activate CTL alone.Methods We utilized a combined approach of using a computer-based algorithm analysis TEPITOPE and in vitro biological analysis to identify Th epitopes in CEA.Results Initial screening of healthy donors showed that all five predicted peptides derived from CEA could induce peptide-specific T-cell proliferation in vitro. We characterized these CEA epitopes by establishing and analyzing peptide-specific T-cell clones. It was shown that CD4+ T-cells specific for the CEA₁₁₆ epitope can recognize and respond to naturally processed CEA protein and CEA₁₁₆ epitope can be promiscuously presented by commonly found major histocompatibility complex (MHC) alleles. Furthermore, it was demonstrated that immunization of human leukocyte antigen (HLA)-DR4 transgenic mice with CEA₁₁₆ peptide elicited antigen-specific Th responses which can recognize the antigenic peptides derived from CEA protein and CEA-positive tumors.Conclusion The MHC class II-restricted epitope CEA₁₁₆ could be used in the design of peptide-based tumor vaccine against several common cancers expressing CEA.     

LEC–BiFC: a new method for rapid assay of protein interaction

Abstract

Protein–protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein–protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC–BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl–X_L and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC–BiFC can be applied easily in any lab equipped with a fluorescence microscope.     

Calpain-resistant fragment(s) of alpha-synuclein regulates the synuclein-cleaving activity of 20S proteasome

Alpha-synuclein is a pathological component of Parkinson's disease by participating in Lewy body formation. Imbalance in protein turnover could result in the abnormal protein aggregation responsible for eventual neuronal cell death. This in vitro digestion study showed that both m-calpain and 20S proteasome preferentially hydrolyzed the N-terminal half of alpha-synuclein, which made the hydrophobic NAC and following acidic C-terminal region resistant against the proteolyses. Since the acidic C-terminal region contains the PEST segment-a protein degradation signal enriched with amino acids of proline (P), glutamate (E), serine (S), and threonine (T)-, the PEST segment has not been processed or even required for the proteolyses. Alpha-synuclein would be recognized primarily by m-calpain since the common substrate was processed by m-calpain five times more effectively than 20S proteasome with k(cat)/K(m) of 1.64 x 10(4)M(-1)s(-1) and 0.32 x 10(4) M(-1)s(-1), respectively. The N-terminally truncated protease-resistant C-terminal fragment of alpha-syn61-140 was demonstrated to stimulate the 20S proteasome-mediated breakdown of alpha-synuclein and its mutant forms of Ala53Thr and Ala30Pro. The stimulation for Ala53Thr, however, was noticeably less efficient than those for the other proteins, which might support the previous observation of the prolonged intracellular life span of Ala53Thr by 1.5-fold compared to that of wild-type form. We have hypothesized that the N-terminally truncated C-terminal fragment derived from the abundant alpha-synuclein through intracellular proteolyses could be involved in various physiological or pathological effects which might be related to the formation of abnormal protein aggregation and subsequent neuronal degeneration by influencing the intracellular protein turnover or directly participating in the aggregate formation.     

Mesangial cell apoptosis induced by a fibronectin fragment

We previously showed that in passive Heymann nephritis (PHN) rats, a large quantity of fibronectin (FN) fragments containing the central cell-binding (CCB) domain and adjacent domains are generated in the kidney and excreted into urine (Nishizawa et al., Biol Pharm Bull 1998; 21: 429–433). To ascertain whether the FN fragments could affect the progression of PHN, we investigated the effect of a 150 K FN fragment containing the CCB and carboxyl-terminal heparin-binding (Hep 2) domains on cultured rat mesangial cells. When rat mesangial cells cultured on FN-coated plates were exposed to the 150 K FN fragment, some mesangial cells detached from the FN substrate and then underwent apoptosis as judged by nuclear and DNA fragmentations. The 150 K FN fragment competitively inhibited the mesangial cell adhesion to the FN substrate in a dose-dependent manner. Furthermore, gelatinzymography of the conditioned medium of mesangial cells showed that the 150 K FN fragment induced and/or poteintiated the extracellular matrix (ECM)-degrading proteinases including matrix metalloproteinases (MMPs) of mesangial cells. These results indicate that the 150 K FN fragment may elicit mesangial cell apoptosis by disrupting the mesangial cell adhesion through two distinct ways: the inhibition of mesangial cell adhesion and the ECM-degradation by the 150 K FN fragment-induced MMPs. Thus, FN fragments containing the CCB and adjacent domains generated in the kidneys of PHN rats may be involved in the evolution of the renal injury.     

Posttranslational Modification of 6-Phosphofructo-1-Kinase in Aspergillus niger

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the V_max of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.     

Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA.

The complete nucleotide sequence of turnip yellow mosaic virus (TYMV) genomic RNA has been determined on a set of overlapping cDNA clones using a sequential sequencing strategy. The RNA is 6318 nucleotides long, excluding the cap structure. The genome organization deduced from the sequence confirms previous results of in vitro translation. A novel open reading frame (ORF) putatively encoding a Pro-rich and very basic 69K (K = kilodalton) protein is detected at the 5' end of the genome. It is initiated at the first AUG codon on the RNA and overlaps the major ORF that encodes the non structural 206K (previously referred to as 195K) protein of TYMV; its function is unknown. Several amino acid consensus sequences already described among plant and animal viruses are also found in the TYMV-encoded polypeptides. A comparison with other viruses whose RNA sequence is known leads to the conclusion that TYMV belongs to the "Sindbis-like" supergroup of viruses and could be related to Semliki forest virus.     

Cell-free synthesis and transport of precursors of mutant ornithine carbamoyltransferases into mitochondria

Synthesis, mitochondrial transport and processing of ornithine carbamoyltrasferase (EC 2.1.3.3) were studied in mutant mice strains (sparse-fur, spf, and sparse-fur with abnormal skin and hair, spf-ash) which exhibit a deficiency in this enzyme. Spf mice have an increased amount (about 150% of control) of the enzyme with abnormal kinetic properties, whereas spf-ash mice have a decreased amount (about 10% of control) of the enzyme with apparently normal kinetic properties. Precursors of the mutant enzymes were synthesized in a reticulocyte lysate cell-free system. The hepatic level of translatable mRNA coding for the enzyme and the rate of the enzyme synthesis in liver slices of spf mice were 58 and 60% of the controls, respectively. In the case of spf-ash mice the activity of translatable mRNA for the enzyme was 10% of the controls. These results indicate that the decreased amount of ornithine carbamoyltransferase protein in spf-ash mice is due mainly to a decreased level of translatable mRNA for the enzyme, whereas the increase in the enzyme amount in spf mice is presumably the result of a decreased rate of enzyme degradation. The subunit molecular weight of the spf enzyme precursor was practically the same as that of the normal enzyme precursor (M_r 40 000). Both precursors synthetized in vitro could be taken up and processed similary to an apparently mature form (M_r 37 000). In the case of spf-ash enzyme, two discrete in vitro products were observed on sodium dodecyl sulfate polyacrylamide gel; one comigrated with the normal enzyme precursor and the other moved slightly slower. Both products appeared to be taken up and processed to the mature form of the enzyme.     

The epidermal growth factor precursor isolated from murine kidney membranes. Chemical characterization and biological properties

To understand the biology and the biochemistry of the epidermal growth factor (EGF) precursor in normal tissues we partially purified the EGF precursor from mouse kidney. The precursor was purified by affinity chromatography, using wheat germ lectin and antibodies to murine EGF. The EGF precursor is a glycosylated integral membrane protein of apparent molecular mass of 140-150 kDa. The solubilized EGF precursor is biologically active as evidenced by its ability to compete with 125I-labeled EGF for binding to the EGF receptor in intact fibroblasts and its ability to stimulate the growth of cells dependent on EGF for growth. The EGF precursor from mouse kidney can be proteolytically processed by the EGF-associated arginine esterase into a smaller fragment (97 kDa) that retains both immunologic sensitivity to EGF antiserum and biological activity. Extensive digestion of the EGF precursor with pepsin liberates a biologically and immunologically active protein of approximately the size of mature EGF.     

Orientation of the cleavage map of the 200-kilodalton polypeptide encoded by the bottom-component RNA of cowpea mosaic virus

The genomic organization of the bottom-component RNA of cowpea mosaic virus was studied. In vivo, this RNA encodes at least eight different polypeptides of 170, 110, 87, 84, 60, 58, 32, and 4 kilodaltons (K), the last polypeptide representing the genome-bound protein VPg. In rabbit reticulocyte lysates, bottom-component RNA is translated into a 200K polypeptide which is then processed to give the 32 and 170K polypeptides also found in vivo. By pulse-labeling the 200K primary translation product, we now show that the 32 and 170K polypeptides are derived from the NH₂-terminal and COOH-terminal parts of this polypeptide, respectively. Comparison of the proteolytic peptide patterns of 170K polypeptides synthesized in vitro and pulse-labeled at either the NH₂-terminal or the COOH-terminal end with the patterns of the 170 and 110K polypeptides found in vivo demonstrates that the order within the 200K primary translation product of cowpea mosaic virus bottom-component RNA is as follows: NH₂-32K polypeptide-58K polypeptide-VPg-24K polypeptide-87K polypeptide-COOH.     

Cleavage of a hydrophilic C-terminal domain increases growth-inhibitory activity of oncostatin M.

Oncostatin M is a polypeptide cytokine, produced by normal and malignant hematopoietic cells, that has several in vitro activities, including the ability to inhibit growth of cultured carcinoma cells. Here we present a structural and functional comparison of two oncostatin M-related proteins (Mr 36,000 and 32,000) secreted by COS cells transfected with oncostatin M cDNA. The smaller of these forms lacked a hydrophilic C-terminal domain comprising predominantly basic amino acids. This domain was also absent from native oncostatin M produced by U937 cells. The 32,000-Mr form of oncostatin M was not produced by cells transfected with plasmids (G195 and G196) in which a potential trypsinlike cleavage site within the hydrophilic C-terminal domain was altered by site-directed mutagenesis. A 32,000-Mr fragment was produced by trypsin treatment of the 36,000-Mr form of oncostatin M. These observations suggest that the 32,000-Mr form of oncostatin M was derived from the 227-amino-acid propeptide by proteolytic cleavage at or near the paired basic residues at positions 195 and 196. Pro-oncostatin M was equally active in radioreceptor assays as the processed form but was 5- to 60-fold less active in growth inhibition assays. Likewise, nonprocessed mutant protein encoded by plasmid G196 was equally active in the radioreceptor assays as the processed form but was five- to ninefold less active in growth inhibition assays. Thus, the highly charged C-terminal domain of pro-oncostatin M is not required for receptor binding or growth-inhibitory activity but may alter the functional properties of the molecule. Propeptide processing of oncostatin M may be important for regulating in vivo activities of this cytokine.     

Proteolytic origin of the 150-kilodalton protein encoded by turnip yellow mosaic virus genomic RNA.

Turnip yellow mosaic virus genomic RNA codes in vitro for two overlapping proteins, 150-kilodalton (150K protein) and 206-kilodalton (206K protein) proteins. The proteolytic maturation known to affect the 206K protein has been further characterized by in vitro translation assays in a reticulocyte lysate or wheat germ extract. Cleavage is inhibited at 37 degrees C and restored when the temperature is shifted to 30 or 25 degrees C. Temperature shift experiments are used here to demonstrate that the 150K protein and the previously characterized 78K protein are the two fragments resulting from a primary cleavage phenomenon that affects the 206K protein in a cotranslational manner under usual translation conditions. This processing is prevented by several cysteine and serine proteinase inhibitors.     

Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber

Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers. It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage. This size difference could not be accounted for. The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned. Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control. Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage. A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO2H-terminal, are removed. Therefore, the protein produced from the truncated gene was larger because it cannot be processed. This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein. The amber codon could be located 24 codons away from the normal stop codon. Obviously, the fragment cannot be processed. The existence of this fragment demonstrates that processing occurs during phage maturation.     

Ultrafast dynamics-driven biomolecular recognition where fast activities dictate slow events

In general, biological macromolecules require significant dynamical freedom to carry out their different functions, including signal transduction, metabolism, catalysis and gene regulation. Effectors (ligands, DNA and external milieu, etc) are considered to function in a purely dynamical manner by selectively stabilizing a specific dynamical state, thereby regulating biological function. In particular, proteins in presence of these effectors can exist in several dynamical states with distinct binding or enzymatic activity. Here, we have reviewed the efficacy of ultrafast fluorescence spectroscopy to monitor the dynamical flexibility of various proteins in presence of different effectors leading to their biological activity. Recent studies demonstrate the potency of a combined approach involving picosecond-resolved Förster resonance energy transfer, polarisation-gated fluorescence and time-dependent stokes shift for the exploration of ultrafast dynamics in biomolecular recognition of various protein molecules. The allosteric protein–protein recognition following differential protein–DNA interaction is shown to be a consequence of some ultrafast segmental motions at the C-terminal of Gal repressor protein dimer with DNA operator sequences O_E and O_I. Differential ultrafast dynamics at the C-terminal of λ-repressor protein with two different operator DNA sequences for the protein–protein interaction with different strengths is also reviewed. We have also systemically briefed the study on the role of ultrafast dynamics of water molecules on the functionality of enzyme proteins α-chymotrypsin and deoxyribonuclease I. The studies on the essential ultrafast dynamics at the active site of the enzyme α-chymotrypsin by using an anthraniloyl fluorescent extrinsic probe covalently attached to the serine-195 residue for the enzymatic activity at homeothermic condition has also been reviewed. Finally, we have highlighted the evidence that a photoinduced dynamical event dictates the molecular recognition of a photochromic ligand, dihydroindolizine with the serine protease α-chymotrypsin and with a liposome (L-α-phosphatidylcholine).     

A carboxyl-terminal fragment of Plasmodium falciparum gp195 expressed by a recombinant baculovirus induces antibodies that completely inhibit parasite growth.

The major merozoite surface Ag (gp195) of Plasmodium falciparum has been shown to protect monkeys against parasite infection, and gp195-based synthetic peptides and recombinant polypeptides have been evaluated as potential malaria vaccines. A major problem in developing a gp195-based recombinant vaccine has been the difficulty in obtaining a recombinant polypeptide that is immunologically equivalent to the native protein. In this study, the carboxyl-terminal processing fragment (p42) of gp195 was produced in yeast and in a baculovirus recombinant system. Immunologic analyses indicated that the secreted baculovirus p42 (BVp42) expressed native, disulfide-dependent conformational epitopes, whereas these epitopes were poorly represented in the intracellular yeast p42. BVp42, but not yeast p42, was also recognized by the majority of gp195-specific antibodies of animals immunized with purified native gp195, indicating that the anti-gp195 response of these animals was focused on conformational determinants of the p42 processing fragment. Sera against native gp195 of congenic mice of diverse H-2 haplotypes recognized the BVp42 polypeptide, demonstrating that a genetically heterogeneous population is capable of responding to p42 epitopes. BVp42 was highly immunogenic and induced high titers of antibodies that were cross-reactive with purified native gp195 in an ELISA and also reacted with schizonts and merozoites by immunofluorescence. Anti-BVp42 antibodies completely inhibited the in vitro growth of the malaria parasite, whereas anti-yeast p42 antibodies had no effect. These results indicate that native, conformational epitopes of p42 are critical for the induction of gp195-specific, parasite growth-inhibitory antibodies and that the BVp42 polypeptide efficiently induces antibodies specific for these native determinants.     

Isolation and biological characterization of active fragments of the adhesive glycoprotein fibronectin

Cellular fibronectin is a major cell surface glycoprotein that can mediate the adhesion of cells to collagen in vitro. To analyze its mechanism of action, we have undertaken experiments to isolate fragments of fibroblast fibronectin that retain different active sites. In this paper, we describe the purification of three chymotryptic fragments with apparent molecular weights of 40,000, 160,000 and 205,000 from chicken cellular fibronectin. These fragments were electrophoretically pure and retained different biologically active sites, as determined by a series of bioassays and competitive inhibition experiments. The 40K fragment was identified as the collagen-binding fragment. The 160K fragment was found to contain the cell surface-binding site(s) of cellular fibronectin. The 205K fragment contained both collagen-binding and cell surface-binding sites, and apparently represents the sum of the 40K and 160K fragments. When native fibronectin is cleaved to the 205K fragment, a polypeptide region containing all interchain disulfide bonds is lost. This alteration was accompanied by decreased hemagglutinating activity and loss of the capacity to restore a normal morphology to transformed cells, whereas cell attachment to collagen and cell spreading activities remained. Our results directly support the idea that the fibronectin molecule consists of separate structural domains containing different biological characteristics.     

Increased expression of ribosome recycling factor is responsible for the enhanced protein synthesis during the late growth phase in an antibiotic-overproducing Streptomyces coelicolor ribosomal rpsL mutant

K88E mutation within rpsL, which encodes the S12 ribosomal protein, enhanced the protein synthetic activity of Streptomyces coelicolor during the late growth phase, resulting in overproduction of the deep blue-pigmented polyketide antibiotic actinorhodin. In vitro cross-mixing experiments using the ribosomal and S-150 fractions derived from wild-type and K88E mutant strains suggested that one or more translation factors are enriched in the mutant's S-150 fraction, while Western analysis using antibodies against various translation factors revealed ribosome recycling factor (RRF) to be one of the enriched mediators. RRF purified from overexpressing cells stimulated mRNA-directed green fluorescent protein (GFP) synthesis in an in vitro protein synthesis system. GFP synthesis rates were complemented by RRF addition into wild-type cell's S-150 fraction, eliminating the difference between wild-type and mutant S-150 fractions. The frr gene encoding RRF was found to be transcribed from two distinct start points (frrp1 and frrp2), and increased expression from frrp1 could account for the elevated level of RRF in the K88E mutant during the late growth phase. Moreover, introduction of a plasmid harbouring a high copy number of frr gene into wild-type S. coelicolor induced remarkable overproduction of antibiotic, demonstrating that the increased levels of RRF caused by the K88E mutation is responsible for an aberrant stationary-phase event: overproduction of antibiotic.     

Pentasaccharide enhances the inactivation of factor Xa by antithrombin by promoting the assembly of a Michaelis-type intermediate complex. Demonstration by rapid kinetic, surface plasmon resonance, and competitive binding studies

It has been demonstrated that a unique pentasaccharide fragment of heparin (H5) activates AT by exposing an exosite on the serpin that is a recognition site for interaction with the basic autolysis loop (residues 143-154) of fXa. In support of this, the substitution of Arg-150 of fXa with Ala (R150A) impaired the reactivity of the mutant with AT by 1 order of magnitude specifically in the presence H5. To understand the mechanism by which heparin activation of AT improves the reactivity of the serpin with fXa, the H5-catalyzed reaction of AT with fXa, fXa R150A, and fXa S195A was studied using rapid kinetic, surface plasmon resonance, and competitive binding methods. The pseudo-first-order rate constants for the H5-catalyzed AT inhibition of both fXa and fXa R150A exhibited a linear dependence on the serpin concentration, thereby yielding second-order rate constants of 1.0 x 10(6) and 1.5 x 10(5) M(-)(1) s(-)(1), respectively. On the other hand, an approximately 70-saccharide, high-affinity heparin-catalyzed AT inhibition of both fXa derivatives showed a saturable dependence on the inhibitor concentration, yielding an identical rate constant of approximately 20 s(-)(1), but different ternary fXa-heparin-AT dissociation constants (K(E,ATH)) of approximately 130 and approximately 1780 nM for wild-type and R150A fXa, respectively. Competitive kinetic and surface plasmon resonance binding studies using the catalytically inactive S195A mutant of fXa yielded dissociation constants of 255 and 610 nM, respectively, for the mutant protease interaction with the AT-H5 complex. These results suggest that H5 enhances the reactivity of AT with fXa primarily by lowering the K(E,ATH) for the formation of a Michaelis-type serpin-protease encounter complex.