Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts

Abstract

Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties.

Probing contacts of inhibitor locked in transition states in the catalytic triad of DENV2 type serine protease and its mutants by 1H, 19F and 15 N NMR spectroscopy

“…In our earlier NMR studies we demonstrated the existence of the above mentioned LBHB between H51 and D75 of the catalytic triad in the transition state of DENV2 NS2B:NS3pro with substrate-analogue boronic acid inhibitor Bz-Nle-Lys-Arg-Arg-B(OH)2, (compound I, Table 1) [20]. To our knowledge, this is the first time the existence of a LBHB type complex in serine proteases, as has been predicted [12], could be demonstrated by NMR spectroscopy in a biological system. The unusual large low-filed shift of N δ1 H (19.93 ppm) of H51 combined with a N-H splitting of only 52 Hz clearly indicated the presence of LBHB [20].…”

“…The exact nature of the hydrogen bonds in the catalytic triad is of importance in order to understand the mechanism. It has been suggested that the aspartate hydrogen bond to the histidine is a delocalized low barrier hydrogen bond (LBHB) [11,12]. LBHB is thus important to the understanding of the structure of the tetrahedral transition state for the functioning enzyme.…”

“…Other parts involved in the protease function are: the peptide binding site and the oxyanion hole which stabilizes the negative charge on a deprotonated oxygen [13]. There are several reports on serine proteases where the interaction between the catalytic triad and boronic or aldehyde substrate analogue inhibitors have been studied and there is an understanding that in different type of serine proteases different mechanisms could prevail involving different modes of inhibitor binding [12][13][14][15][16][17][18]. Remarkably, in the x-ray structure of West Nile virus (WNV) NS2B:NS3pro with a short peptide boronic acid type of inhibitor recently obtained the inhibitor orientation in the active site is complemented by interaction with an additional molecule, glycerol, present in the enzyme [19].…”

1-(3-Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM/MM Studies

“…QM/MM optimization of non-covalent complex of hAChE with TFK in the active site obtained by molecular docking leads to size expansion of the active site necessary to accommodate the inhibitor and lower energy barrier for formation of covalent adduct. Traditionally, resulting hemiketals are called "transition state analogs", though practically they are rather analogs of tetrahedral intermediate of AChE-catalyzed ester hydrolysis [55,56]. Hemiketal configuration is very close for TFK and TMTFA, with the only difference that trimethyl ammonium group of TMTFA is 0.6 Å closer to Glu202 than tert-butyl moiety of TMTFA.…”

Peptidyl Fluoromethyl Ketones and Their Applications in Medicinal Chemistry

“…These two fluorinated moieties exhibit another important characteristic; they can easily hydrate, depending on the nature of the amino acids they are bound to, forming very stable gem -diols which mimic the tetrahedral adduct of the transition-state of the enzyme-substrate hydrolysis mechanism ( Figure 1 ). Therefore, the resulting PFMKs may also act exclusively as transition-state competitive analogues leading to rapidly reversible competitive inhibition [ 5 , 6 ]. In most cases, a mixture of the two FMK forms (i.e., carbonyl and hydrated) in different ratio is observed in solution, and a dual inhibition mechanism of the PFMKs is detected consequently.…”