Differences in Three Kinetic Parameters Underpin the Unique Catalytic Profiles of Nitric-oxide Synthases I, II, and III

Abstract

We previously reported the existence of a special autoregulation 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 (San- The free radical nitric oxide (NO) 1 is involved in an increasing number of physiologic and pathophysiologic processes (1-6). NO is generated by the NO synthases, which are found in numerous organisms. Animal NO synthases are homodimers whose subunits are comprised of a reductase domain containing FAD, FMN, and NADPH binding sites, and an oxygenase domain containing 6(R)-tetrahydrobiopterin (H 4 B), iron protoporphyrin IX (heme), and a binding site for the L-arginine (Arg) substrate (7,8). NO synthases catalyze two sequential mixedfunction oxidations, the first being Arg hydroxylation to form N -hydroxy-L-arginine (NOHA) as a bound intermediate (9 -11), and the second converting NOHA to citrulline and NO.toliniThree main NO synthases are expressed in mammals and differ in their functions, amino acid sequence, post-translational modification, and cellular location. Two NOS, neuronal NOS (nNOS or NOS-1) and endothelial NOS (eNOS or NOS-3), are constitutively expressed and involved in signal cascades. The third NOS is cytokine-inducible (iNOS or NOS-2) and functions as both a regulator and effector of the immune response. The counterpart to this diversity of location and function is a specific regulation of each isoform. For example, NO synthases differ significantly regarding Ca 2ϩ levels required to bind calmodulin (CaM), which triggers heme reduction and NO synthesis (12, 13). They also have different capacities to be upor down-regulated by serine/threonine phosphorylation (14, 15). A third difference involves regulation via heme⅐NO complex formation. In nNOS, we have established that heme⅐NO complex formation is an intrinsic feature that governs the rate of NO synthesis and shifts the enzyme apparent K m O 2 to a higher value (16,17). The model shown in Scheme 1 below can explain these effects (18). It incorporates the observation that ferric heme binds newly formed NO before it can leave the enzyme (19,20). This causes nNOS to partition between futile and productive cycles during catalysis (see Scheme 1), with only the productive cycle liberating NO (18). Key kinetic parameters for nNOS have been measured, including rates of heme reduction and NO dissociation, k cat , and oxidation of the ferrous heme⅐NO complex (19,21,22). These values are set such that a majority of nNOS exists as the ferrous heme⅐NO complex during steady-state NO synthesis (23,24). Computer simulation of the kinetic model in Scheme 1 reproduces this result and can accurately simulate the pre-steady-state and steady-state behaviors of nNOS mutants that display greater or diminished activity relative to wild-type enzyme (18,25).The available data for iNOS and eNOS suggest r...

A Conserved Val to Ile Switch near the Heme Pocket of Animal and Bacterial Nitric-oxide Synthases Helps Determine Their Distinct Catalytic Profiles

“…6. The slower k off values have typically been derived from equilibrium NO titration experiments (25,26,40), in ligand displacement studies (16,25,26) or in single turnover catalytic studies (13,14,22,25,26) that directly follow NO dissociation from the ferric heme as we have done here in Figs. 3-5.…”

“…1). In the three mammalian NOS, the rates of Fe III NO dissociation and reduction are set so that much of the NO can escape from the enzyme before the Fe III NO product complex is reduced to ferrous, which then dooms it to an oxidative reaction that forms a higher oxide of nitrogen in place of NO (15,16). The Fe III NO dissociation rates of mammalian NOS enzymes are within a range that is typical for other ferric heme proteins, whereas in bsNOS this rate is near the lower end of the range (Table V).…”

“…1). Interestingly, there is only a modest variation in Fe III NO dissociation rates among the three mammalian NOS isoforms, which range from 2 to 5 s Ϫ1 when measured in NOHA single turnover reactions at 10°C (16).…”

“…Because dissociation of the Fe III NO product complex is required for NO to escape from the enzyme, it is an essential step for the biologic functions of animal NOSs. In fact, this dissociation rate is one of three key kinetic parameters that together determine the overall catalytic behavior of a given NOS (15,16). Slower rates of NO dissociation dispose the Fe III NO product complex toward its reduction during catalysis, which then places the ferrous heme-NO enzyme species into a futile cycle that does not release NO (Fig.…”

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…[31]). It includes transcriptional [32], translational and posttranslational [33][34][35] regulations of NO-synthesis mechanisms, negative feedback regulation of NOS by NO [36][37][38][39], membrane transport of substrates [40][41][42][43], intracellular metabolic recycling of substrates [43][44][45][46][47][48][49][50][51], availability of molecular oxygen and cell respiration [52][53][54][55][56][57], and intracellular redox state [58][59][60].…”