Substrate specificity of recombinant human renal renin: effect of histidine in the P2 subsite of pH dependence

Green, David W.; Aykent, Serdar; Gierse, James K.; Zupec, Mark E.

doi:10.1021/bi00464a032

Cited by 40 publications

(30 citation statements)

References 35 publications

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“…4B and Table I). This is an unprecedented finding for aspartyl proteases, because modest and normal kinetic isotope effects on k cat have been reported for HIV protease (10), renin (41), and pepsin (12). The slow step in these cases was associated with the "recharging" of the enzyme after bond breaking, most likely reflecting enzyme reprotonation to the initial state and/or regaining of the catalytic water.…”

Section: Discussionmentioning

confidence: 74%

Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

Toulokhonova¹,

Metzler²,

Witmer³

et al. 2003

Journal of Biological Chemistry

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The steady-state kinetic mechanism of ␤-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine-(Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k cat /K m , a large inverse solvent kinetic isotope effect on k cat , and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the ratelimiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at ␦ 13.0 ppm and of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k cat . Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K ii and K is expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of ␤-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.Extracellular amyloid deposits in brain, a characteristic feature of Alzheimer's disease, is a result of proteolytic cleavage of membrane-bound amyloid precursor protein by two enzymes, ␤-secretase and ␥-secretase. The second cleavage activity (␥-secretase) is strongly associated with the presenilin multisubunit complexes (1), whereas ␤-secretase (BACE) 1 has been identified as a novel transmembrane aspartyl protease (2-4).Although aspartyl proteases have been studied for more than 4 decades, new aspects of catalysis and inhibition continue to emerge. A substantial number of these enzymes have been identified as useful targets for chemotherapeutic intervention in human diseases (5-8), yet there has been limited success in identifying clinically relevant inhibitors; hence, it is important to explo...

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Section: Discussionmentioning

confidence: 74%

Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

Toulokhonova¹,

Metzler²,

Witmer³

et al. 2003

Journal of Biological Chemistry

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“…This conclusion is based on three-dimensional structural data of these enzymes, affinity labeling studies, and the observation of “bell-shaped” profiles of log V/K vs pH or log V vs. pH for oligopeptide substrates of porcine pepsin, 40, 41, 42, 43 penicillopepsin, 44 and human renin. 45 Moreover, this statement was recently confirmed by Kuroki and co-workers, 46 who observed a proton on only one aspartate residue using neutron diffraction technique. The resulting structural data showed that the catalytic residue Asp25(A) is protonated and that Asp25(B) is deprotonated.…”

Section: Introductionmentioning

confidence: 68%

Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease

2016

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HIV-1 Protease (HIV-1 PR) is one of the three enzymes essential for the replication process of HIV-1 virus, which explains why it has been the main target for design of drugs against acquired immunodeficiency syndrome (AIDS). This work is focused on exploring the proteolysis reaction catalyzed by HIV-1 PR, with special attention to the dynamic and electrostatic effects governing its catalytic power. Free energy surfaces for all possible mechanisms have been computed in terms of potentials of mean force (PMFs) within hybrid QM/MM potentials, with the QM sub-set of atoms described at semiempirical (AM1) and DFT (M06-2X) level. The results suggest that the most favorable reaction mechanism involves formation of a gem-diol intermediate, whose decomposition into the product complex would correspond to the rate-limiting step. The agreement between the activation free energy of this step with experimental data, as well as kinetic isotope effects (KIEs), supports this prediction. The role of the protein dynamic was studied by protein isotope labelling in the framework of the Variational Transition State Theory. The predicted enzyme KIEs, also very close to the values measured experimentally, reveal a measurable but small dynamic effect. Our calculations show how the contribution of dynamic effects to the effective activation free energy appears to be below 1 kcal·mol−1. On the contrary, the electric field created by the protein in the active site of the enzyme emerges as being critical for the electronic reorganization required during the reaction. These electrostatic properties of the active site could be used as a mould for future drug design.

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“…CD36 was isolated and identified as a platelet integral membrane glycoprotein (Green et al, 1990; Greenwalt et al, 1992). It also goes by the name of FAT (fatty acid translocase) because it can bind long-chain free fatty acids and transport them into cells (Febbraio et al, 2001; Silverstein and Febbraio, 2009; Silverstein et al, 2010).…”

Section: Basic Science Of the Macular Pigment Carotenoidsmentioning

confidence: 99%

Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease

Bernstein

Vachali

et al. 2016

Progress in Retinal and Eye Research

435

419

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The human macula uniquely concentrates three carotenoids: lutein, zeaxanthin, and meso-zeaxanthin. Lutein and zeaxanthin must be obtained from dietary sources such as green leafy vegetables and orange and yellow fruits and vegetables, while meso-zeaxanthin is rarely found in diet and is believed to be formed at the macula by metabolic transformations of ingested carotenoids. Epidemiological studies and large-scale clinical trials such as AREDS2 have brought attention to the potential ocular health and functional benefits of these three xanthophyll carotenoids consumed through the diet or supplements, but the basic science and clinical research underlying recommendations for nutritional interventions against age-related macular degeneration and other eye diseases are underappreciated by clinicians and vision researchers alike. In this review article, we first examine the chemistry, biophysics, and physiology of these yellow pigments that are specifically concentrated in the macula lutea through the means of high-affinity binding proteins and specialized transport and metabolic proteins where they play important roles as short-wavelength (blue) light-absorbers and localized, efficient antioxidants in a region at high risk for light-induced oxidative stress. Next, we turn to clinical evidence supporting functional benefits of these carotenoids in normal eyes and for their potential protective actions against ocular disease from infancy to old age.

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Substrate specificity of recombinant human renal renin: effect of histidine in the P2 subsite of pH dependence

Cited by 40 publications

References 35 publications

Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease

Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease

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