Formation Of Encounter Complex Research Articles

Rapid-reaction Kinetic Characterization of the Pathway of Streptokinase-Plasmin Catalytic Complex Formation

Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys(414) binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK.Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys(414) deletion mutant (SKDeltaK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKDeltaK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKDeltaK414 was approximately 10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK.Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys(414) binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes.

Mechanism and Energy Landscape of Domain Swapping in the B1 Domain of Protein G

Three-dimensional domain swapping has emerged as a ubiquitous process for homo-oligomer formation in many unrelated proteins, but the molecular mechanism of this process is still poorly understood. Here we present a mechanism for the swapping reaction in the B1 domain of the immunoglobulin G binding protein from group G of Streptococcus (GB1). This is a particularly attractive system for investigating the swapping process, as the swapped dimer formed by the quadruple mutant (L5V/F30V/Y33F/A34F) of GB1 was recently shown to exist in equilibrium with a monomer-like conformation over time scales of minutes. According to our mechanism, swapping in GB1 starts from the C-terminus of the polypeptide chain and progresses by exchanging an increasing portion of the chains until a stable conformational state is reached. This exchange process does not involve unfolding. Rather, the conformational changes of individual monomers and their association are tightly coupled to minimize solvent exposure and maximize the total number of native contacts at all times, thereby closely approximating the minimum energy path of the reaction. Using detailed atomic descriptions, we compute the complete free-energy profiles of the exchange reaction for the GB1 quadruple mutant that forms swapped dimers and for the wild-type protein, which is monomeric. In both GB1 forms, intermediates sample a surprisingly wide range of nearly isoenergetic association modes and hinge conformations, indicating that the exchange reaction is a non-specific process akin to encounter complex formation where the amino acid sequence plays a marginal role. The main role of the mutations in the swapping process is to destabilize the GB1 monomer state, while stabilizing the swapped dimer conformation, with non-native intersubunit interactions, fostered by mutant side chains, contributing significantly to this stabilization. Our findings are rationalized in terms of a generic swapping mechanism that involves the association of activated molecular species, and it is argued that a similar mechanism may apply to swapping in other protein systems.

Binding-Induced Folding of a Natively Unstructured Transcription Factor

Transcription factors are central components of the intracellular regulatory networks that control gene expression. An increasingly recognized phenomenon among human transcription factors is the formation of structure upon target binding. Here, we study the folding and binding of the pKID domain of CREB to the KIX domain of the co-activator CBP. Our simulations of a topology-based Gō-type model predict a coupled folding and binding mechanism, and the existence of partially bound intermediates. From transition-path and Φ-value analyses, we find that the binding transition state resembles the unstructured state in solution, implying that CREB becomes structured only after committing to binding. A change of structure following binding is reminiscent of an induced-fit mechanism and contrasts with models in which binding occurs to pre-structured conformations that exist in the unbound state at equilibrium. Interestingly, increasing the amount of structure in the unbound pKID reduces the rate of binding, suggesting a “fly-casting”-like process. We find that the inclusion of attractive non-native interactions results in the formation of non-specific encounter complexes that enhance the on-rate of binding, but do not significantly change the binding mechanism. Our study helps explain how being unstructured can confer an advantage in protein target recognition. The simulations are in general agreement with the results of a recently reported nuclear magnetic resonance study, and aid in the interpretation of the experimental binding kinetics.

Effect of surface potential of reverse micelle on enzyme–substrate complex formation

The influence of the electrostatic potential of reverse micelle on the enzyme–substrate complex formation have been studied within the framework of the encounter complex (EC) formation theory. Reverse micelles have a multiple-factor effect on encapsulated substances and on mechanisms of their interaction. The action of individual constituents on the overall micellar effect was analyzed by means of Brownian dynamics simulation. The effects of surface potential in charged and neutral reverse micelles, of the size and of the ionic strength of micellar core on the probability to form the reaction complex between serine protease (trypsin) and specific substrate Nα-benzoyl- l -arginine ethyl ester was examined. It was shown that negative potential of micelle increases the probability of EC formation and the positive potential decreases it. Orientation of substrate in electrostatic field of micelle depends on the value and the sign of surface potential.

Exosites in the substrate specificity of blood coagulation reactions.

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Restraining the conformation of HIV-1 gp120 by removing a flexible loop

The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.

Estimation of Electronic Coupling for Intermolecular Electron Transfer from Cross-Reaction Data

Sixty-five electron-transfer reactions including 27 new 0, +1 couples have been added to our data set of cross-reactions between 0 and +1 couples, bringing it to 206 reactions involving 72 couples that have been studied by stopped-flow kinetics in acetonitrile containing supporting electrolyte at 25 degrees C, formal potentials determined by cyclic voltammetry, and analyzed using Marcus cross-rate theory. Perhaps surprisingly, a least-squares analysis demonstrates that intrinsic rate constants exist that predict the cross-rate constants to within a factor of 2 of the observed ones for 93% of the reactions studied, and only three of the reactions have a cross-rate constant that lies outside of the factor of 3, that corresponds to a factor of 10 uncertainty in the rate constant for an unknown couple. Many triarylamines, which have very high intrinsic reactivity, are included among the newly studied couples. The enthalpy contribution to the Marcus reorganization energy, lambda'v, has been calculated for 46 of the couples studied, at the (U)B3LYP/6-31+G (or for the larger and lower barrier compounds, at the less time-consuming (U)B3LYP/6-31G) level. In combination with a modified Levich and Dogodnadze treatment that assumes that the rate constant is proportional to (KeHab2/lambda1/2) exp[-DeltaG/RT], this allows estimation of the electronic coupling (Hab) at the transition state for intermolecular electron transfer, (more properly H'ab, the product of the square root of the encounter complex formation constant times Hab) for these couples. Although the principal factor affecting intermolecular electron-transfer rate constants is clearly lambda, H'ab effects are easily detectable, and the dynamic range in our estimates of them is over a factor of 600.

Polypeptide binding proteins: what remains to be discovered?

Many proteins have developed the potential to sequester a client polypeptide chain in its various folding states as a specific intermolecular ligand and, thus, exhibit the properties of a holding chaperone. The resulting complexes can be of a diverse nature in terms of structure and reaction dynamics and are characterized on the basis of various microscopic properties including formation and decay of encounter and Michaelis complexes as well as reactant and product stability. Interpretation of the functional consequences of complex formation in the cell generally tends to be rather complicated, with notable exceptions including complexes formed during the reaction pathways of proteases, protein kinases and protein phosphatases. Peptide bond cis/trans isomerases take up an intermediate position among the poly(oligo)peptide binding proteins because, although the relationship between chain sequestration and catalysis of isomerization can easily be delineated in vitro, it is sometimes difficult to resolve in the cell. Time-resolved studies on interactions involving peptide bond cis/trans isomerases have led to the establishment of generally applicable methods for studying protein-poly(oligo)peptide interactions that are capable of identifying new types of biocatalysis.

Lysozyme dimerization: brownian dynamics simulation

The lysozyme dimerization reaction has been studied within the framework of encounter-complex (EC) formation theory using the MacroDox software package. Two types of energetically favorite ECs were determined. In the first of them, active-center amino acids of lysozyme take part in the complex formation or the second molecule blocks accessibility to active center sterically. Epitope amino-acid residues are involved in the complex of type II. The existence of both types of complexes does not contradict experimental data. Dimer-formation rate constants for different kinds of EC were calculated. Increasing the pH from 2.0 to 10.0 decreases the total positive lysozyme charge and eliminates the unfavorable repulsive electrostatic interaction. The rate constant of EC formation is inversely proportional to the protein total charge. The association rate constant was also enhanced by an increase of ionic strength that screened repulsive electrostatic interaction between positively charged proteins.

Electron transfer reaction in the chromium(VI)-manganese(II) system in the presence of ethylenediaminetetraacetic acid (EDTA)

Upon addition of Cr VI to a solution of ethylenediaminetetraacetic acid (EDTA) and Mn II, a transient species appears which has an absorption maximum at 500 nm. Kinetic studies of the outer-sphere oxidation of the Mn II-EDTA complex with the Cr VI-EDTA complex have been investigated by visible spectrophotometry at 25 °C. The formation of a transient species has been characterized spectrophotometrically and the encounter complex formation constants have been determined (KOS = 1.75 × 102 and 1.66 × 103 mol-2 dm6 for [EDTA] and [MnII] variations, respectively). The effect of total [EDTA], [MnII], [Cr VI] and [HClO4] on the rate of the reaction was determined. On addition of HClO4, there is a decrease in the rate constants. The reaction product is the CrIII-EDTA complex with λmax = 400 and 550 nm. On the basis of the various observations and product characterization a most plausible outer-sphere mechanism has been envisaged.

Do enzymes bind their substrates in the ground state because of a physico-chemical requirement?

Transition state theory has provided no convincing explanation for the nearly universal observation of complexes of enzymes with substrates. Bimolecular catalytic reactions are assumed here to take place through reactive encounter complexes defined as the subset of reactant state species able to proceed directly to low lying energy levels at the transition state. By assessing the probability of these complexes from the maximum efficiency of intramolecular reactions, an upper limit for the rate constants promoted by hypothetical catalysts unable to bind substrates is deduced. This limit, which is below the ordinary range of bimolecular rate constants ( k cat/ K M) for enzyme reactions, results from a kinetic limitation in the formation of reactive encounter complexes. Exceeding this limit requires a stabilization of these complexes. Using the terminology of transition state theory, the need for enzymes to form complexes with substrates is then expressed as a necessity to restore Boltzmann distribution at the transition state.

Solvent and salt effects on the kinetics of the reaction between [Ru(NH3)5pz]2+ and [Fe(CN)5H2O]3–

AbstractThe kinetics of replacement of H2O by [Ru(NH3)pz]2+ (pz = pyrazine) in [Fe(CN)5H2O]3− have been studied in various concentrated electrolyte solutions and in various water–cosolvent mixtures, at 298 K. Salt and cosolvent effects can be rationalized taking into account specific medium effects on both the encounter complex formation process and the ligand‐substitution process, once the encounter complex is formed. These effects in water–cosolvent mixtures depend on the solvation of the reactants by the components of the mixture, as well as on the solvent–solvent interactions in these mixtures. Salt effects seem to be related to a primary salt effect as well as to the effect of the cations on the electronic density on the iron complex. © 2003 Wiley Periodicals, Inc. Int J Chem Kinet 35: 367–373, 2003

Photochemistry of methoxyhydroquinone and methoxy-p-benzoquinone in solution related to the photoyellowing of the lignocellulosics.

The photoreactivity of methoxy-p-benzoquinone (MQ) and methoxyhydroquinone (MHQ) in dilute solution (10(-4)-10(-3) M) was studied using continuous irradiation and laser flash photolysis (LFP). The quinone irradiated in degassed tetrahydrofuran (THF) gives MHQ and an adduct with the solvent. Only the formation of hydroquinone is observed in ethanol, and hydroxylation is evidenced in water, whereas the compound is stable in CCl4. The bis-quinone, 4,4'-dimethoxybiphenyl-2,5,2',5'-bisquinone, and the dibenzofurane-quinone, 8-hydroxy-3,7-dimethoxydibenzofuran-1,4-quinone, are formed in the presence of MHQ, whereas the reactivity is low with ethylconiferyl alcohol. When MHQ is irradiated selectively in degassed THF, the formation of MQ and of the bis-hydroquinone, 4,4'-dimethoxy-2,5,2',5'-tetrahydroxy-biphenyl, are observed. The dimer is oxidized photochemically or thermally into the mono- or bis-quinones, the process being accelerated in alkaline medium. The formation of the dimers is strongly favored by the contiguous presence of quinone and hydroquinone. When MHQ is selectively irradiated in the presence of transethylconiferaldehyde (EtC), quinone formation and isomerization of EtC are observed. LFP experiments, performed with a selective excitation of MQ, indicate that the triplet state of the quinone is strongly quenched by MHQ to conduce to a semiquinone radical. The interaction between 3MQ* and MQ is mainly driven by an electron transfer process according to the similar value of the quenching rate constant found with another electron donor compound such as 1,4-dimethoxybenzene. By contrast, no strong quenching of 3EtC* by MHQ was observed. It is proposed that the photochemistry of the couple MQ/MHQ is governed by the formation of encounter complex between either 3MQ* and MHQ or 3MHQ* and MQ. Consequently, the fast part of the photoyellowing of lignocellulosics does not appear to involve the couple MHQ/MQ or MHQ/etherified coniferaldehyde, but more likely a combination of oxidation of the hydroquinone by ground-state oxygen and photohydration of the formed quinone from its triplet state, giving inter-alia more colored o-quinonoid type molecules.

Steric Control of Electron Transfer. Changeover from Outer-Sphere to Inner-Sphere Mechanisms in Arene/Quinone Redox Pairs

The various aromatic hydrocarbons (Chart 2) constitute a sharply graded series of sterically encumbered (unhindered, partially hindered, and heavily hindered) donors in electron transfer (ET) to quinones (Chart 1). As such, steric effects provide the quantitative basis to modulate (and differentiate) outer-sphere and inner-sphere pathways provided by matched pairs of hindered and unhindered donors with otherwise identical electron-transfer properties. Thus the hindered donors are characterized by (a) bimolecular rate constants (k2) that are temperature dependent and well correlated by Marcus theory, (b) no evidence for the formation of (discrete) encounter complexes, (c) high dependency on solvent polarity, and (d) enhanced sensitivity to kinetic salt effectsall diagnostic of outer-sphere electron-transfer mechanisms. Contrastingly, the analogous unhindered donors are characterized by (a) temperature-independent rate constants (k2) that are 102 times faster and rather poorly correlated by Marcus theory, (b) weak dependency on solvent polarity, and (c) low sensitivity to kinetic salt effectsall symptomatic of inner-sphere ET mechanisms arising from the preequilibrium formation of encounter complexes with charge-transfer (inner-sphere) character. Steric encumbrances which inhibit strong electronic (charge-transfer) coupling between the benzenoid and quinonoid π systems are critical for the mechanistic changeover. Thus, the classical outer-sphere/inner-sphere distinction (historically based on coordination complexes) is retained in a modified form to provide a common terminology for inorganic as well as organic (and biochemical) redox systems.

Kinetic epitope mapping of the chicken lysozyme.HyHEL-10 Fab complex: delineation of docking trajectories.

The rate constants, k(on), for the formation of hen (chicken) lysozyme (HEWL). Fab-10 complexes have been determined for wild-type (WT) and epitope-mutated lysozymes by a homogeneous solution method based on the 95% reduced enzymatic activity of the complex. The values fall within a narrow 10-fold range [(0.18 to 1.92) x 10(6) M(-1)s(-l)]. The affinity constants, K(D), cover a broader, 440-fold, range from 0.075 to 33 nM. Values of K(D) as high as 7 microM were obtained for the complexes prepared from some mutations at HEWL positions 96 and 97, but the associated kinetic constants could not be determined. The values of k(on) are negatively correlated with side-chain volume at position 101HEWL, but are essentially independent of this parameter for position 21HEWL substitutions. The multiple mutations made at positions 21HEWL and 101HEWL provide sufficient experimental data on complex formation to evaluate phi values [phi = (deltadeltaGon)/(deltadeltaG(D))] at these two positions to begin to define trajectories for protein-protein association. The data, when interpreted within the concept of a two-step association sequence embracing a metastable encounter complex intermediate, argue that the rate determining step at position 21HEWL (phiavg = 0.2) is encounter complex formation, but the larger phi(avg) value of 0.36 experienced for most position 101HEWL mutations indicates a larger contribution from the post-encounter annealing process at this site for these replacements.

Brownian Dynamics Simulation of Protein–Protein Diffusional Encounter

Protein association events are ubiquitous in biological systems. Some protein associations and subsequent responses are diffusion controlledin vivo.Hence, it is important to be able to compute bimolecular diffusional association rates for proteins. The Brownian dynamics simulation methodology may be used to simulate protein–protein encounter, compute association rates, and examine their dependence on protein mutation and the nature of the physical environment (e.g., as a function of ionic strength or viscosity). Here, the theory for Brownian dynamics simulations is described, and important methodological aspects, particularly pertaining to the correct modeling of electrostatic forces and definition of encounter complex formation, are highlighted. To illustrate application of the method, simulations of the diffusional encounter of the extracellular ribonuclease, barnase, and its intracellular inhibitor, barstar, are described. This shows how experimental rates for a series of mutants and the dependence of rates on ionic strength can be reproduced well by Brownian dynamics simulations. Potential future uses of the Brownian dynamics method for investigating protein–protein association are discussed.

Simulation of the diffusional association of barnase and barstar

The rate of protein association places an upper limit on the response time due to protein interactions, which, under certain circumstances, can be diffusion-controlled. Simulations of model proteins show that diffusion-limited association rates are approximately 10(6)-10(7) M-1 s-1 in the absence of long-range forces (Northrup, S. H., and H. P. Erickson. 1992. Kinetics of protein-protein association explained by Brownian dynamics computer simulations. Proc. Natl. Acad. Sci. U.S.A. 89:3338-3342). The measured association rates of barnase and barstar are 10(8)-10(9) M-1 s-1 at 50 mM ionic strength, and depend on ionic strength (Schreiber, G., and A. R. Fersht. 1996. Rapid, electrostatically assisted association of proteins. Nat. Struct. Biol. 3:427-431), implying that their association is electrostatically facilitated. We report Brownian dynamics simulations of the diffusional association of barnase and barstar to compute association rates and their dependence on ionic strength and protein mutation. Crucial to the ability to reproduce experimental rates is the definition of encounter complex formation at the endpoint of diffusional motion. Simple definitions, such as a required root mean square (RMS) distance to the fully bound position, fail to explain the large influence of some mutations on association rates. Good agreement with experiments could be obtained if satisfaction of two intermolecular residue contacts was required for encounter complex formation. In the encounter complexes, barstar tends to be shifted from its position in the bound complex toward the guanine-binding loop on barnase.

Solvent Effects on the Formation and Decay of an Exciplex between the Lowest Excited Singlet State of 9,10-Dibromoanthracene and Ground-State Amine (N,N-Dimethylaniline or Triethylamine)

Picosecond laser photolysis reveals the formation of an exciplex between the lowest excited singlet state [DBA*(S1)] of 9,10-dibromoanthracene (DBA) and ground-state amine in acetonitrile (CH3CN) and ethanol (EtOH) containing N,N-dimethylaniline (DMA) [and n-heptane (HP) containing DMA or triethylamine (TEA)]. Only in CH3CN−DMA, however, can decomposition of the DBA−DMA exciplex into the DBA radical anion (DBA•-) and the DMA radical cation be seen, indicating very small generation of DBA•- from the DBA−amine exciplex formed in EtOH−DMA and HP−amine (DMA or TEA). Interestingly, no DBA−TEA exciplex is formed in CH3CN and EtOH containing TEA but nanosecond laser photolysis reveals the existence of DBA•- in the former solvent. Furthermore, the rate of DBA → 9-bromoanthracene debromination upon steady-state photolysis in CH3CN−TEA is 1 order of magnitude smaller than that in CH3CN−DMA but 2 or 3 orders of magnitude greater than those in EtOH and HP containing amine. This suggests that diffusion-controlled quenching of DBA*(S1) by TEA in CH3CN and EtOH gives rise to the formation of a nonemissive short-lived encounter complex or ion pair. It thus be concluded that generation of DBA•- from the DBA−amine exciplex or the encounter complex (or the ion pair) is affected by the dielectric constant of a pure solvent.

Quenching of substituted naphthalenes fluorescence by chloromethanes

The fluorescence quenching of some substituted naphthalenes by chloromethanes such as carbontetrachloride (CCl 4), chloroform (CHCl 3) and dichloromethane (CH 2Cl 2) have been studied in a large number of solvents in room temper ature. The quenching rate constants ( k q ) have been determined for all the fluorophores in all the solvents. For CCl 4 quenching the k q depends on solvent viscosity whereas for CHCl 3 and CH 2C1 2 it depends neither on solvent viscosity nor on solvent polarity. From the results the quenching mechanism is explained in terms of an encounter complex formation for CCl 4 quenching and an intermediate donor-acceptor complex formation for CHCl 3 and CH 2Cl 2 quenching in the excited state.

Kinetic Characterization of the Proteinase Binding Defect in a Reactive Site Variant of the Serpin, Antithrombin: ROLE OF THE P1′ RESIDUE IN TRANSITION-STATE STABILIZATION OF ANTITHROMBIN-PROTEINASE COMPLEX FORMATION

To elucidate the role of the P1' residue of the serpin, antithrombin (AT), in proteinase inhibition, the source of the functional defect in a natural Ser-394-->Leu variant, AT-Denver, was investigated. AT-Denver inhibited thrombin, Factor IXa, plasmin, and Factor Xa with second order rate constants that were 430-, 120-, 40-, and 7-fold slower, respectively, than those of native AT, consistent with an altered specificity of the variant inhibitor for its target proteinases. AT-Denver inhibited thrombin and Factor Xa with nearly equimolar stoichiometries and formed SDS-stable complexes with these proteinases, indicating that the diminished inhibitor activity was not due to an enhanced turnover of the inhibitor as a substrate. Binding and kinetic studies showed that heparin binding to AT-Denver as well as heparin accelerations of AT-Denver-proteinase reactions were normal, consistent with the P1' mutation not affecting the heparin activation mechanism. Resolution of the two-step reaction of AT-Denver with thrombin revealed that the majority of the defective function was localized in the second reaction step and resulted from a 190-fold decreased rate constant for conversion of a noncovalent proteinase-inhibitor encounter complex to a stable, covalent complex. Little or no effects of the mutation on the binding constant for encounter complex formation or on the rate constant for stable complex dissociation were evident. These results support a role for the P1' residue of antithrombin in transition-state stabilization of a substrate-like attack of the proteinase on the inhibitor-reactive bond following the formation of a proteinase-inhibitor encounter complex but prior to the conformational change leading to the trapping of proteinase in a stable, covalent complex. Such a role indicates that the P1' residue does not contribute to thermodynamic stabilization of AT-proteinase complexes and instead favors a kinetic stabilization of these complexes by a suicide substrate reaction mechanism.