Previous studies of dopamine beta-monooxygenase (D beta M) have implicated the formation of a substrate-derived benzylic radical via a hydrogen atom abstraction mechanism [Miller & Klinman (1985) Biochemistry 24, 2114]. We now address the nature of the oxygen species catalyzing C-H bond cleavage through the measurement of oxygen-18 isotope effects as a function of substrate structure. Using deuterium isotope effects, together with experimental O-18 isotope effects with protonated and deuterated substrates, it has been possible to calculate intrinsic O-18 isotope effects. Since the D beta M mechanism includes many steps which may involve changes in bond order at dioxygen, e.g., the reversible binding of O2 to the active-site copper and its reductive activation to a copper-hydroperoxide species, the intrinsic O-18 isotope effect is expected to be the product of two terms: (1) an overall equilibrium O-18 isotope effect on steps leading from O2 binding to the formation of the intermediate which catalyzes C-H bond cleavage and (2) a kinetic O-18 isotope effect on the C-H bond cleavage step. Thus, the magnitude of a single O-18 isotope effect measurement cannot reveal the nature of the bonding at oxygen during substrate activation. In the present study we have measured the change in O-18 isotope effect as a function of substrate structure and reactivity, finding values of 18(V/K) which decrease from 1.0281 +/- 0.001 to 1.0216 +/- 0.0003 as the rate of the C-H bond cleavage step decreases from 680 to 2 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral deposition of amyloid ␤-protein (A␤) is believed to play a key role in the pathogenesis of Alzheimer's disease. Because A␤ is produced from the processing of amyloid ␤-protein precursor (APP) by ␤-and ␥-secretases, these enzymes are considered important therapeutic targets for identification of drugs to treat Alzheimer's disease. Unlike ␤-secretase, which is a monomeric aspartyl protease, ␥-secretase activity resides as part of a membrane-bound, high molecular weight, macromolecular complex. Pepstatin and L685458 are among several structural classes of ␥-secretase inhibitors identified so far. These compounds possess a hydroxyethylene dipeptide isostere of aspartyl protease transition state analogs, suggesting ␥-secretase may be an aspartyl protease. However, the mechanism of inhibition of ␥-secretase by pepstatin and L685458 has not been elucidated. In this study, we report that pepstatin A methylester and L685458 unexpectedly displayed linear non-competitive inhibition of ␥-secretase. Sulfonamides and benzodiazepines, which do not resemble transition state analogs of aspartyl proteases, also displayed potent, non-competitive inhibition of ␥-secretase. Models to rationalize how transition state analogs inhibit their targets by non-competitive inhibition are discussed. Accumulation and deposition of ␤-amyloid (A␤)1 peptides in the cerebral cortex is believed to be an early and central process in the pathogenesis of Alzheimer's disease. The A␤ peptides are generated from sequential proteolytic cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases, which are therefore considered important targets for therapeutic intervention. Molecular cloning (1-3) and crystallographic studies (4) have unequivocally established ␤-secretase as an aspartyl protease. However, the identity of ␥-secretase remains elusive.It is known that transmembrane proteins presenilin 1 (PS1) and presenilin 2 (PS2) are essential for intramembranous proteolytic ␥-cleavage of APP (5) and a few other ␥-secretase substrates such as Notch (6 -9) and ErbB4 (10). Evidence suggests that presenilins may have direct catalytic activity (11, 12), but recent reports indicate that this activity requires interactions between presenilins and other proteins such as nicastrin (13) and co-fractionates with a very high molecular weight complex (14). Mature presenilins themselves form subunit heterodimers between the N-and C-terminal fragments, which are generated from endoproteolytic cleavage of the full-length presenilin (15, 16). This complex membrane-bound molecular organization has hindered efforts to purify and reconstitute ␥-secretase activity.In the absence of purified enzyme and crystal structures, inhibition studies have played a prominent role in the understanding of the nature of ␥-secretase. ␥-secretase activity is sensitive to aspartyl protease transition state analogs such as the hydroxyl ethylene isosteres, pepstatin (17-19) and L685458 (20), typical aspartyl protease transition state inhibitors. Peptidomimetics containing a dif...

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Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.

Tyrosine hydroxylase converts tyrosine to dihydroxyphenylalanine utilizing a tetrahydropterin cofactor and molecular oxygen. Previous deuterium isotope effect studies have raised the possibility that the activation of oxygen might be the rate-limiting step for this reaction. To test the validity of this proposal, we have measured the 18O kinetic isotope effects for the tyrosine hydroxylase reaction as a function of amino acid substrate, tetrahydropterin derivative, and pH. The measured 18O isotope effects are nearly constant in every condition tested with an average value of 1.0175 ± 0.0019. These results are consistent with a change in the bond order to oxygen in the rate determining step. Furthermore, the isotope effects measured with the coupled substrate 4-methoxyphenylalanine and the completely uncoupled substrate 4-aminophenylalanine are identical, implying the same rate determining step independent of whether oxygen activation is coupled to substrate hydroxylation. The results of these studies provide strong support for a rate limiting reductive activation of molecular oxygen, most likely via a one-electron transfer from the tetrahydropterin to form superoxide anion as the first reactive intermediate.

The delta H associated with the thermal unfolding of G-actin has been determined by differential scanning calorimetry (DSC) to be 142 +/- 5 kcal/mol, with the Tm (melting temperature) at 57.2 +/- 0.5 degrees C, at pH 8.0 (heating rate 0.5 K/min). The transition is broad and cannot be treated as a single transition that mimics a two-state process, suggesting the existence of domains. Deconvolution is done to fit it into two quasi-independent two-state transitions. For F-actin, the transition is more cooperative, with a cooperative ratio (the ratio of van't Hoff enthalpy and calorimetric enthalpy) of 1.4, indicating intermonomer interaction. The delta H of the thermal unfolding of F-actin is 162 +/- 10 kcal/mol with a Tm at 67.0 +/- 0.5 degrees C. A state of G-actin similar to that of the heat-denatured form, designated D-actin, is obtained by removing tightly bound Ca2+ with EGTA. The DSC-detectable cooperative transition is completely lost when the free calcium concentration of the medium is 1 x 10(-11) M or lower, using a Ca2+/EGTA buffer system. However, circular dichroism (CD) shows that the helix content of actin, 32% in the G-form, is only partially reduced to 19% in this apo form. The CD spectrum and the helix content of the calcium-depleted actin are almost identical with those of the heat-denatured D form. This loss of 40% of the native helical content is irreversible in both cases. The remaining 60% of the native helical content cannot be further eliminated by heating to 95 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

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Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS)