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Enzymatic Degradation of Bradykinin
Published in Sami I. Said, Proinflammatory and Antiinflammatory Peptides, 2020
Randal A. Skidgel, Ervin G. Erdös
Carboxypeptidase M has a neutral pH optimum and cleaves only C-terminal Arg or Lys from a variety of synthetic peptide substrates as well as naturally occurring peptides such as Bk (Fig. 1), Arg6- and Lys6-enkephalins, and dynorphin A1–13 (Skidgel et al., 1989). Carboxypeptidase M cleaves C-terminal arginine preferentially over lysine, and the penultimate residue can prominently affect the rate of hydrolysis. Thus, Met5-Arg6-enkephalin has the highest specificity constant (kc|Km = 20.3μM-1min-1), and changing the C-terminal amino acid to lysine (Met5-Lys6-enkephalin) decreases it over 10-fold due to a large increase in the Km (Skidgel et al., 1989). Although the Km of Leu5-Arg6-enkephalin (63 μM) is similar to that of Met5-Arg6-enkephalin (46 μM), the kcat is almost ninefold lower, indicating the effect of the penultimate residue on hydrolysis. Of the substrates tested, Bk (with C-terminal Phe8-Arg9) has the lowest Km (16 μM) and the second highest kcat|Km (9.2 μM -1 min-1) (Table 1).
Peptidases and Peptides at the Blood-Brain Barrier
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
Janet Brownlees, Carvell Williams
Although the physiological substrates for DPP IV in vivo have not as yet been identified, the enzyme has been shown to cleave a range of peptides and proteins in vitro, some of which are present in the brain microvasculature. Generally, small peptides are degraded much more rapidly than proteins although Km values seem to be independent of the peptide chain length.179 Thus the specificity constant, Vm/Km, declines with increasing size of the peptide. The unique cyclic structure of the imino acid proline not only influences the conformation of peptide chains, but also restricts the action of most proteases, even those of broad specificity. The physiological role of DPP IV may therefore be to cleave peptides and proteins containing proline residues at positions which render them resistant to attack by other peptidases.
Enzyme Kinetics and Drugs as Enzyme Inhibitors
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
k–1 and k1 are the rate constants for the association/dissociation equilibrium between E and S and the ES complex and the Michaelis–Menten constant is given by (k–1 + kcat)/k1. From Vmax in the above Eq. which is the product of kcat and the total enzyme concentration (Et) present, kcat results as Vmax/(Et) which is the total turnover number, the number of substrate molecules converted to the product P presupposed the enzyme molecules are fully saturated with the substrate. The ratio of kcat/KM (in s–1∙ M–1) is taken as a measure of the catalytic efficiency η (also termed specificity constant). For example, chymotrypsin favors the conversion of bulky peptide substrates; for those with phenylalanine h is about 105, for glycine this value is just 1.3 × 10–1 (Fersht, 1999).
Flavin-containing monooxygenase 3 (FMO3): genetic variants and their consequences for drug metabolism and disease
Published in Xenobiotica, 2020
Ian R. Phillips, Elizabeth A. Shephard
The unusual mechanism of FMOs accounts for their broad substrate range. The slowest (rate-limiting) steps in the catalytic cycle are the breakdown of FADH-OH to release H2O and the release of NADP+ (Figure 1, steps 4 and 5). As both occur after the oxygenation of substrate and release of oxygenated product (Figure 1, step 3), the catalytic constant (kcat) is usually independent of the structure of the substrate and the specificity constant (kcat/KM) is determined largely by the KM for the substrate. As FMOs do not form classical Michaelis–Menten enzyme–substrate complexes, KM is a measure of the ease with which a substrate can gain access to the active site. Factors that restrict access to the active site are size, shape and charge; the best substrates are uncharged or have a single positive charge (Poulsen & Ziegler, 1995; Ziegler, 1993, 2002). The catalytic cycle can also undergo uncoupling, producing H2O2 with the release of NADP+ (Siddens et al., 2014) (Figure 1), enabling FMOs to moonlight as NADPH oxidases. The rate of uncoupling is higher in the presence of substrate, with as much as 30–50% of bound oxygen being released as H2O2 (Siddens et al., 2014).
Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Stane Pajk, Damijan Knez, Urban Košak, Maja Zorović, Xavier Brazzolotto, Nicolas Coquelle, Florian Nachon, Jacques-Philippe Colletier, Marko Živin, Jure Stojan, Stanislav Gobec
The progress-curve analysis was performed using the ENZO web application, implemented at www.enzo.cmm.ki.si20–22. This programme is designed to generate differential equations from drawn reaction schemes and subsequently to fit the coefficients of these equations according to least squares methodology to reproduce the experimental data using a numerical integration algorithm. For determination of the kinetics mechanisms and parameters, the van Slyke-Cullen single intermediate reaction scheme23 for substrate hydrolysis by huBChE was combined with a reversible mixed inhibition mechanism (Figure 1). In all of the evaluations, the k1 of 814 s−1, the substrate specificity constant k1/Km of 1.0 × 108 M−1−1s–t, and the inhibition constant of 54 µM for the binding of the thiocholine-TNB (k3/k2) in the hydrolysis of BSCh by purified huBChE were constrained, as these values had been determined previously24.
Leishmania infantum arginase: biochemical characterization and inhibition by naturally occurring phenolic substances
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Andreza R. Garcia, Danielle M. P. Oliveira, Ana Claudia F. Amaral, Jéssica B. Jesus, Ana Carolina Rennó Sodero, Alessandra M. T. Souza, Claudiu T. Supuran, Alane B. Vermelho, Igor A. Rodrigues, Anderson S. Pinheiro
ARGLi exhibited classical Michaelis–Menten kinetics, a hyperbolic dependency of the reaction rate as a function of L-arginine concentration. Non-linear regression of the Michaelis–Menten plot enabled the determination of kinetic parameters, such as Vmax (0.28 ± 0.016 mM/min) and Km (5.1 ± 1.1 mM) (Figure 2(C)). ARGLi Km values are more similar to those described for human arginase I (Km=7.6 mM)35 than for native and recombinant L. amazonensis arginase (Km=23.9 ± 0.96 and 21.5 ± 0.9, respectively)12, recombinant L. mexicana arginase (Km=25 ± 4 mM)11, and recombinant human arginase (Km=13 ± 2 mM)11. Our results suggest that ARGLi shows a stronger affinity for L-arginine than arginase from human and tegumentary Leishmania species. From the Km and Vmax values, we calculated a Kcat of 2.55 × 103 s−1 and a specificity constant (Kcat/Km) of 5 × 108 M−1s−1. The high Kcat and Kcat/Km values suggest that ARGLi displays excellent catalytic efficiency, higher than that described for L. mexicana arginase (Kcat=1.7 s−1)11.