Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects

Rebholz, Karen L.; Northrop, Dexter B.

doi:10.1016/0006-291x(91)90890-j

Cited by 23 publications

(34 citation statements)

References 9 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%

“…After protonation of the leaving amine and collapse of the tetrahedral intermediate (step 4 in Scheme 1), the enzyme returns to its original protonation state (species GH in the Scheme 1) but still lacks the catalytic water molecule. It has been proposed that reprotonation or rehydration of the enzyme active site (step 7 in Scheme 1) may be a rate-limiting step during pepsin (12) and HIV protease catalysis (10).…”

Section: Discussionmentioning

confidence: 99%

“…Kinetic mechanism studies suggest that pepsin and HIV protease undergo isomerization steps during catalysis (10,11), and some evidence was obtained that this iso-step might be rate-limiting (12). Inhibitors binding to the form of the enzyme that accumulates because of the slow interconversion between enzyme isomers would be expected to display higher affinity and greater selectivity for the target enzyme.…”

mentioning

confidence: 99%

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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%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…porcine pepsin) (Polgár, 1989). It has been reported that a water molecule attacks the carbonyl carbon of the scissile bond, serving as a third catalytic component along with the active aspartic carboxyl groups, in the catalysis by aspartic proteases (Rebholz & Northrop, 1991); two catalytically active aspartate residues are in either a right or wrong protonic state, involving general basecatalyzed attack by a water molecule on the carbonyl carbon of the scissile bond. Recent abinitio molecular dynamics simulations on HIV-1 protease were focused on the catalytic D 25 and D 25/ , and resolved all the uncertainties within a unifying hypothesis, as illustrated in figure 5 (Piana & Carloni, 2000).…”

Section: Catalytic Motifs and Sequences Of Two Three Etc Subsitesmentioning

confidence: 99%

“…Thereupon, it seems as more accurate that serine as well as cysteine proteases perform catalysis not by means of the routinely known catalytic triad and or dyad but through extraordinary manipulation of up to five catalytic residues. A similar treatment is worth for aspartic proteases and metalloproteases (Auld, 1997;Rawlings & Barret, 1994;Rebholz & Northrop, 1991;Svensson, 1994) showing that in general, the viewing of an enzymatic mechanism should not be focused only on the markedly referred catalytic residues. The concept of the catalytic sequence gets of special interest, when we have to do with -amylase-like enzymes as it is illustrated in figures 3(a) and (b), where what extraordinary happens is that each monomer of polymeric substrates acts as catalytic residue!…”

Section: Catalytic Motifs and Sequences Of Two Three Etc Subsitesmentioning

confidence: 99%

Effective Kinetic Methods and Tools in Investigating the Mechanism of Action of Specific Hydrolases

Papamichael¹,

Stergiou²,

Foukis³

et al. 2012

Medicinal Chemistry and Drug Design

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A Conserved Lysine in β‐Lactam Synthetase Assists Ring Cyclization: Implications for Clavam and Carbapenem Biosynthesis

et al. 2009

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β-Lactam synthetase (β-LS) is the paradigm of a growing class of enzymes that form the critical β-lactam ring in the clavam and carbapenem antibiotics. β-LS catalyzes a two stage reaction in which N2-(2-carboxyethyl)-l-arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to β-lactam formation is high, and that the overall rate of reaction is partially limited to a protein conformational change rather than to the chemical step alone of closing the strained ring. β-Lactam formation was evaluated on the basis of X-ray crystal structures, site-specific mutation, and kinetic and computational studies. The combined evidence clearly points to a reaction coordinate involving the formation of a tetrahedral transition state/intermediate stabilized by a conserved Lys. The combination of substrate pre-organization, a well-stabilized transition state and an excellent leaving group facilitates this acyl substitution to account for the strong forward commitment to catalysis and to lower the barrier of four-membered ring formation to the magnitude of a protein conformational change.

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Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects

Cited by 23 publications

References 9 publications

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

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

Effective Kinetic Methods and Tools in Investigating the Mechanism of Action of Specific Hydrolases

A Conserved Lysine in β‐Lactam Synthetase Assists Ring Cyclization: Implications for Clavam and Carbapenem Biosynthesis

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