Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å ß barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

Application of the second rule of transient-state kinetic isotope effects to an enzymatic mechanism.

The transient-state kinetic approach reveals the formation and subsequent interconversions of intermediates in real time. Its potential for the mechanistic resolution of enzymatic and other complex chemical mechanisms has been severely limited however by the lack of a rigorous and applicable theoretical basis in contrast to that of the less direct but soundly based algebraic algorithms of the steady-state approach. Having recently established three rigorously derived fundamental "rules" of transient-state kinetics applicable to realistic multiple step reactions, we present here the successful application of the very counterintuitive "second rule" to the resolution of the mechanism of the l-phenylalanine dehydrogenase catalyzed reaction.

The interpretation of multiple-step transient-state kinetic isotope effects.

In contrast to steady-state kinetic isotope effects (KIEs), transient-state tKIEs are both time and signal dependent and therefore require a very different form of theory for their interpretation. We have previously provided such a theory for the case of single-step isotopic substitutions. No such properly derived theory applicable to the analysis of multiple-step isotopic substitutions required by transient-state solvent isotope effect studies has been available up to this time. Here, we set forth a more general form of that theory which is applicable to multiple-step substituted cases. We prove three theorems: 1. the observed transient-state KIE for any given reactive component in the reaction sequence evaluated at zero time (tKIE(0)) is in fact the arithmetic product of the intrinsic KIEs of all the steps that precede the formation of that component. 2. The observed tKIE(0) is completely independent of the intrinsic KIEs of any reverse step in the reaction. 3. The intrinsic KIE of any step may be obtained by dividing the value of the tKIE(0) for that step by the value of the tKIE(0) of the immediately preceding step in the reaction sequence.

The direct measurement of thermodynamic parameters of reactive transient intermediates of the L-glutamate dehydrogenase reaction.

In a previous report (Fisher, H. F., Maniscalco, S. J., and Tally, J. (2002) Biochem. Biophys. Res. Commun. 287, 343-347) we demonstrated the capability of the "Le Chatelier forcing method" of producing stable solutions containing substantial amounts of transitory enzyme intermediate complexes that can otherwise be observed only fleetingly in the millisecond time range. The method requires nothing more than running an enzyme reaction using forcing concentrations of reactants against an equally forcing concentration of products until equilibrium is attained. Here we have applied this approach to the measurement of the thermodynamics of several such reactive (and normally transient) intermediate complexes of the bovine liver l-glutamate dehydrogenase-catalyzed reaction. At pH 9.5 and 20 degrees C, we observe both the enzyme-NADPH-alpha-iminoglutarate and enzyme-NADPH-alpha-carbinolamine complexes at concentrations whose sum accounts for 70% of the total enzyme. The pH dependence of these two complexes under equilibrium conditions provides thermodynamic parameters for both the protonated and the unprotonated forms of each of these two entities as well as those of the enzyme-NADP-l-glutamate complex. The equilibrium concentrations of each of these reactive complexes are compared with their corresponding transient steady-state values.

Detection of multiple active site domain motions in transient-state component time courses of the Clostridium symbiosum L-glutamate dehydrogenase-catalyzed oxidative deamination reaction.

We present a multiwavelength, transient-state kinetic study of the oxidative deamination reaction catalyzed by Clostridium symbiosum glutamate dehydrogenase (csGDH) producing the real-time reaction courses of spectroscopically resolved kinetically competent intermediate complexes. The results show striking differences from a corresponding transient-state study of the same reaction by the structurally homologous enzyme from beef liver (blGDH). In addition to the highly blue-shifted alpha-iminoglutarate and highly red-shifted carbinolamine complexes observed in both reactions, the csGDH reaction appeared to show the release of free NADH at a very early and mechanistically unlikely point in the reaction. Using lactic acid dehydrogenase as a "reporter" for free NADH, we show that the early portion of this signal reflects previously unobserved spectrally unshifted enzyme-bound NADH complexes. We provide experimental evidence to show that such spectrally anomalous complexes must represent forms of the known alpha-imino and alpha-carbinolamine complexes in which the active site cleft is open. This evidence includes isothermal calorimetric measurements and pH-jump experiments that show the existence of differing two-state transitions in blGDH and csGDH and locate active site domain motions at differing points in the transient-state time courses of the two enzyme reactions. We prove the kinetic competence of a new and more highly detailed mechanism for the csGDH reaction that involves the alternation of open and closed enzyme complexes as integral steps. These findings, supported by the available X-ray crystal structure data, suggest the existence of a programmed time course of protein domain motions coordinated with the classically considered chemical time course. This new viewpoint may be presumed to be applicable to enzyme reactions other than those of the alpha-amino acid dehydrogenases.