The freely diffusible and moderately reactive free radical, nitric oxide (NO), 2 is a biological signal molecule in numerous physiological and pathophysiological processes (for reviews, see Refs. 1-4). Nitric-oxide synthases (NOSs) catalyze the NADPH-dependent conversion of L-arginine to NO and L-citrulline (for reviews, see Refs. 5-7). In mammals, three different isoforms have been identified. Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, and their activities are Ca 2ϩ /CaM-dependent, whereas the inducible NOS (iNOS) is independent of intracellular Ca 2ϩ concentration. These isoforms share ϳ55% sequence identity yet differ in their size, tissue distribution, and regulation. The 165-kDa nNOS is located in neurons in the brain and neuromuscular junctions and is involved in neurotransmission. eNOS has a molecular mass of 133 kDa, is located in vascular endothelial cells, and is involved in vascular homeostasis. iNOS can be found in macrophages and many other tissues, has a molecular mass of 130 kDa, and is expressed only in response to endotoxins or inflammatory cytokines.All three isoforms of NOS are modular, homodimeric hemoflavoproteins. The N-terminal half of each NOS isozyme is similar to the cytochrome P450 enzyme family and contains iron protoporphyrin IX (heme). It is referred to as the heme domain or the oxygenase domain. This latter domain also contains tetrahydrobiopterin-and arginine-binding sites. The C-terminal half of each isozyme is the flavin-binding domain (or reductase domain) and contains FAD-, FMN-, and NADPH-binding sites, much the same as in NADPH-cytochrome P450 oxidoreductase (CYPOR). These two domains are linked by a CaM-binding region (8). The constitutive isoforms (nNOS and eNOS) are Ca 2ϩ -dependent due to their reversible binding of CaM, providing a mechanism for rapid response in a signaling cascade. On the other hand, iNOS has tightly bound Ca 2ϩ /CaM and is virtually independent of Ca 2ϩ concentration (9). In contrast to the other NOS isozymes, it is regulated at the transcriptional level. As in the case of the P450 (CYP)-CYPOR system, the FAD in the reductase domain accepts a pair of electrons in the form of a hydride ion from NADPH and transfers them one at a time to FMN. FMN, in turn, transfers the electrons again one by one to the heme of the other monomer in the NOS dimer (10 -12). However, the mechanisms of electron transfer and regulation of the FMN domain interactions with its electron acceptor (the heme domain) in NOS and related enzymes, including CYPOR and methionine synthase reductase, are largely unknown. Only recently, studies on this subject have been emerging (13)(14)(15)(16).CaM regulates a wide range of cellular functions through its reversible Ca 2ϩ -dependent binding to target proteins, including NOS. CaM regulates NOS activity by controlling the rates of electron transfer between the two flavin cofactors and between * This work was supported, in whole or in part, by National Institutes of Health Grant GM52682 (to J. J. K.
The formation of carbon-carbon bonds via an acyl-enzyme intermediate plays a central role in fatty acid, polyketide, and isoprenoid biosynthesis. Uniquely among condensing enzymes, 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) catalyzes the formation of a carbon-carbon bond by activating the methyl group of an acetylated cysteine. This reaction is essential in Gram-positive bacteria, and represents the first committed step in human cholesterol biosynthesis. Reaction kinetics, isotope exchange, and mass spectroscopy suggest surprisingly that HMGS is able to catalyze the ''backwards'' reaction in solution, where HMG-CoA is cleaved to form acetoacetyl-CoA (AcAc-CoA) and acetate. Here, we trap a complex of acetylated HMGS from Staphylococcus aureus and bound acetoacetyl-CoA by cryo-cooling enzyme crystals at three different times during the course of its back-reaction with its physiological product (HMG-CoA). This nonphysiological ''backwards'' reaction is used to understand the details of the physiological reaction with regards to individual residues involved in catalysis and substrate͞product binding. The structures suggest that an active-site glutamic acid (Glu-79) acts as a general base both in the condensation between acetoacetyl-CoA and the acetylated enzyme, and the hydrolytic release of HMGCoA from the enzyme. The ability to trap this enzyme-intermediate complex may suggest a role for protein dynamics and the interplay between protomers during the normal course of catalysis. H MGS (3-hydroxy-3-methylglutaryl-CoA synthase) catalyzes the condensation of acetoacetyl-CoA (AcAc-CoA) and acetyl-CoA (Ac-CoA) to form 3-hydroxy-3-methylglutaryl (HMG)-CoA in the first committed, transcriptionally regulated step in cholesterol and isoprenoid biosynthesis. The cholesterol biosynthetic pathway is a proven target for the regulation of serum cholesterol (1, 2), and the mevalonate pathway is essential for many Gram-positive bacteria (3). As described in detail elsewhere (4), the overall three-dimensional structure of HMGS provides structural evidence that the enzyme is a member of the thiolase-like fold (condensing enzyme) superfamily. Mechanistically, the condensation reaction catalyzed by HMGS is reminiscent of the condensation reaction catalyzed by the fatty acid and polyketide condensing enzymes thiolase, -keto-acyl carrier protein synthases, and chalcone synthase. However, in the second (condensation) step of the reaction, HMGS and the HMGS-like polyketide synthesizing enzymes (e.g., JamH; ref. 5) are distinct in that the methyl group of the acetylated enzyme is activated and attacks the incoming -keto thioester, whereas the remaining members of the family activate a carbon on the second substrate to attack the enzyme-bound thioester. Overall, the reaction proceeds by means of a ping-pong mechanism as delineated in Scheme 1 (6). It has been thought that the reaction is irreversible because equilibrium greatly favors the products. On the enzyme, the first step in the reverse reaction is that the carboxylic acid...
Previous work on HMG-CoA synthase has implied the presence of a reactive active site histidine, prompting our examination of the possible function of invariant histidine residues by site-directed mutagenesis. Mutations encoding H197N, H264N/A, and H436N HMG-CoA synthases were constructed, and the mutant enzymes were overexpressed in Escherichia coli BL21(DE3). Kinetic characterization of the isolated synthase variants indicates that, while H197N and H436N enzymes behave similarly to wild-type synthase, H264N and H264A synthases exhibit significant differences. Although the k(m) for acetyl-CoA is not substantially altered, H264N/A synthases catalyze production of HMG-CoA at a diminished (approximately 25-fold slower) rate. In contrast, H264N/A synthases can efficiently catalyze the acetyl-CoA hydrolysis partial reaction exhibiting a k(m) for acetyl-CoA that, again, approximates the value obtained with the wild-type enzyme. These mutants also retain the ability to form significant levels of the acetyl-S-enzyme reaction intermediate. The functional catalysis of partial reactions argues that the H264 mutant proteins retain substantial structural integrity. In this context, it appears significant that the H264N/A synthases exhibit a approximately 100-fold increase in the k(m) for acetoacetyl-CoA. In order to test whether the two orders of magnitude effect may be largely attributed to a decreased affinity of acetoacetyl-CoA for these enzymes and, more specifically, whether H264 interacts with the carbonyl oxygen of acetoacetyl-CoA's thioester, turnover of S-(3-oxobutyl)-CoA, a thioether analog of acetoacetyl-CoA, was investigated. This alternative substrate, in which a methylene group replaces the thioester carbonyl, is utilized by wild-type synthase with an apparent Vmax that is approximately 100-fold lower and an apparent k(m) that is 25-fold higher than the values obtained using the physiological substrate, acetoacetyl-CoA. H264A synthase also catalyzes the turnover of S-(3-oxobutyl)-CoA; the diminution in rate supported by the alternative substrate is comparable in magnitude to the effect observed for wild-type enzyme. In contrast, H264A exhibits comparable apparent k(m) values for S-(3-oxobutyl)-CoA and acetoacetyl-CoA. Thus, unlike wild-type synthase, there is no penalty in terms of efficiency of H264A saturation when the alternative thioether substrate replaces the physiological substrate. These data suggest that the imidazole of H264 in avian enzyme may play a role in anchoring the second substrate, acetoacetyl-CoA, by interacting with the carbonyl oxygen of the thioester functionality.
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