Differences in three kinetic parameters underpin the unique catalytic profiles of nitric-oxide synthases I, II, and III.

We previously reported the existence of a special auto-regulation property of neuronal nitric-oxide synthase (NOS) based on NO near-geminate combination and partial trapping of neuronal NOS (nNOS) through a futile regenerating pathway. On this basis, we developed a kinetic simulation model that was proven to predict nNOS catalytic specificities and mutations effects (Santolini, J., Adak, S., Curran, C. M., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 1233-1243; Adak, S., Santolini, J., Tikunova, S., Wang, Q., Johnson, J. D., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 1244-1252). Here we show that the same model simulates and explains the distinct catalytic behaviors of inducible and endothelial NOS (iNOS and eNOS). Their marked differences were linked to variations in three basic parameters (rates of ferric heme reduction, ferric heme.NO dissociation, and ferrous heme.NO oxidation) that together control partitioning between futile and productive pathways and their relative rates. We also incorporated feedback inhibition into the kinetic model to account for potential rebinding of accumulated solution NO. The model accurately simulated the different relative impacts of both NOS.NO interactions (near-geminate combination of NO versus rebinding of solution NO) on catalytic behavior of each NOS isoform, including their speed and extent of heme.NO complex accumulation, K(m) for O(2), and propensity to transform NO into a higher oxide. Thus, individual catalytic behavior of any NOS can be understood through a single unified kinetic model. Because the model defines how different settings of individual kinetic parameters control regulation by two distinct NOS.NO interactions, it sheds light on mechanisms, structural features, and scope of NOS regulation and its physiologic impact.

Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis.

Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.

A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase.

After initiating NO synthesis a majority of neuronal NO synthase (nNOS) quickly partitions into a ferrous heme-NO complex. This down-regulates activity and increases enzyme K(m,O(2)). To understand this process, we developed a 10-step kinetic model in which the ferric heme-NO enzyme forms as the immediate product of catalysis, and then partitions between NO dissociation versus reduction to a ferrous heme-NO complex. Rate constants used for the model were derived from recent literature or were determined here. Computer simulations of the model precisely described both pre-steady and steady-state features of nNOS catalysis, including NADPH consumption and NO production, buildup of a heme-NO complex, changes between pre-steady and steady-state rates, and the change in enzyme K(m,O(2)) in the presence or absence of NO synthesis. The model also correctly simulated the catalytic features of nNOS mutants W409F and W409Y, which are hyperactive and display less heme-NO complex formation in the steady state. Model simulations showed how the rate of heme reduction influences several features of nNOS catalysis, including populations of NO-bound versus NO-free enzyme in the steady state and the rate of NO synthesis. The simulation predicts that there is an optimum rate of heme reduction that is close to the measured rate in nNOS. Ratio between NADPH consumption and NO synthesis is also predicted to increase with faster heme reduction. Our kinetic model is an accurate and versatile tool for understanding catalytic behavior and will provide new perspectives on NOS regulation.

Kinetic analysis of tentoxin binding to chloroplast F1-ATPase. A model for the overactivation process.

The mechanism of action of tentoxin on the soluble part (chloroplast F1 H+-ATPase; CF1) of chloroplast ATP synthase was analyzed in the light of new kinetic and equilibrium experiments. Investigations were done regarding the functional state of the enzyme (activation, bound nucleotide, catalytic turnover). Dialysis and binding data, obtained with 14C-tentoxin, fully confirmed the existence of two tentoxin binding sites of distinct dissociation constants consistent with the observed Kinhibition and Koveractivation. This strongly supports a two-site model of tentoxin action on CF1. Kinetic and thermodynamic parameters of tentoxin binding to the first site (Ki = 10 nM; kon = 4.7 x 10(4) s-1.M-1) were determined from time-resolved activity assays. Tentoxin binding to the high affinity site was found independent on the catalytic state of the enzyme. The analysis of the kinetics of tentoxin binding on the low affinity site of the enzyme showed strong evidence for an interaction between this site and the nucleotide binding sites and revealed a complex relationship between the catalytic state and the reactivation process. New catalytic states of CF1 devoid of epsilon-subunit were detected: a transient overstimulated state, and a dead end complex unable to bind a second tentoxin molecule. Our experiments led to a kinetic model for the reactivation phenomenon for which rate constants were determined. The implications of this model are discussed in relation to the previous mechanistic hypotheses on the effect of tentoxin.

Interrelation between high and low affinity tentoxin binding sites in chloroplast F1-ATPase revealed by synthetic analogues.

Eight synthetic analogues of tentoxin (cyclo-(L-N-MeGlu1-L-Leu2-N-MeDeltaZPhe3-Gly4)) modified in residues 1, 2, and 3 were checked for their ability to inhibit and reactivate the ATPase activity of the activated soluble part of chloroplast ATP synthase. The data were consistent with a model involving two binding sites of different affinities for the toxins. The occupancy of the high affinity site (or tight site) gave rise to an inactive complex, whereas filling both sites (tight + loose) gave rise to a complex of variable activity, dependent on the toxin analogue. Competition experiments between tentoxin and nonreactivating analogues allowed discrimination between the absence of binding and a nonproductive binding to the site of lower affinity (or loose site). The affinity for the loose site was not affected significantly by the modifications of the tentoxin molecule, whereas the affinity for the tight site was found notably changed. Increasing the size of side chain 1 or 2 and introducing a net electrical charge both resulted in a decrease of affinity for the tight site, but the second change dominated the first one. The activity of different ternary complexes enzyme-tentoxin-analogue depended on the nature of the toxin bound on each site and not only on that bound on the loose site. This demonstrates that the reactivation process results from an interaction, direct or not, between these two binding sites. Possible molecular mechanisms are discussed.

Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.