Kinetics of H2O2-driven catalysis by a lytic polysaccharide monooxygenase from the fungus Trichoderma reesei

Owing to their ability to break glycosidic bonds in recalcitrant crystalline polysaccharides such as cellulose, the catalysis effected by lytic polysaccharide monooxygenases (LPMOs) is of major interest. Kinetics of these reductant-dependent, monocopper enzymes is complicated by the insoluble nature of the cellulose substrate and parallel, enzyme-dependent, and enzyme-independent side reactions between the reductant and oxygen-containing cosubstrates. Here, we provide kinetic characterization of cellulose peroxygenase (oxidative cleavage of glycosidic bonds in cellulose) and reductant peroxidase (oxidation of the reductant) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei. The catalytic efficiency (kcat/Km(H2O2)) of the cellulose peroxygenase reaction (k_cat = 8.5 s⁻¹, and Km(H2O2)=30μM) was an order of magnitude higher than that of the reductant (ascorbic acid) peroxidase reaction. The turnover of H₂O₂ in the ascorbic acid peroxidase reaction followed the ping-pong mechanism and led to irreversible inactivation of the enzyme with a probability of 0.0072. Using theoretical analysis, we suggest a relationship between the half-life of LPMO, the values of kinetic parameters, and the concentrations of the reactants.     

Physicochemical Properties and Amino Acid Composition of Alkaline Proteinase from Aspergillus sojae

Some physicochemical properties and amino acid composition of alkaline proteinase from Aspergillus sojae were found to be as follows: The isoelectric point was at pH 5.1. The molecular weight was 25,500 using the Sheraga-Mandelkern’s formula, based upon the values of the sedimentation coefficient $(s_{20,w}^{°}=\text{?}2.82\text{?}\text{S})$, the intrinsic viscosity ([η] = 0.027 dl/g), and the partial specific volume $({\displaystyle \overline{V}}\text{?}=\text{?}0.726\text{?}\text{ml}/\text{g})$. The enzyme contains 16.8% of nitrogen and is composed of 250 residues of amino acid; Asp₃₁ Glu₁₉, Gly₂₇, Ala₃₂, Val₁₈, Leu₁₄, Ile₁₄, Ser₂₈, Thr₁₈, ${\text{(}{\displaystyle \stackrel{}{{\displaystyle \stackrel{?}{\text{Cys C}}}}}\text{ys})}_1$, Met₂, Pro₆, Phe₇, Tyr₈, Trp₂, His₅, Lys₁₄, Arg₃, (amide-NH₃)₂₀.     

New chemistry based on reduction of homobinuclear bis(μ-phosphido) metal complexes

Studies are reported on the chemical reduction of the homobinuclear bis(μ-phosphido) metal complexes ${(CO)}_3\stackrel{︹}{Fe{(\mu -P{R}_2)}_{2}F}e{(CO)}_3$ (R = Ph or Me), ${(NO)}_2-\stackrel{︹}{Fe{(\mu -PP{h}_2)}_{2}F}e{(NO)}_2$ and ${(CO)}_4\stackrel{︹}{M{(\mu -PP{h}_2)}_{2}M}{(CO)}_4$ (M = Mo or W). Two reduction pathways have been observed which result in different two-electron transformations: (1) with Na or LiAlH₄, electron transfer to yield the corresponding symmetric dianions of the type L_nM(μ-PR₂)₂ML_n^2? without metalmetal bond and (2) with M′BR′₃H(M′ = Li, Na, or K; R′ = Et or sec-Bu), hydride transfer to give monoanionic complexes of the type $L_n\stackrel{︹}{M(\mu -P{R}_2)(\mu -L)M}{L}_{n?1}{(P{R}_{2}H)}^{?}$ or $L_n\stackrel{︹}{M(\mu -P{R}_2)M}{L}_n{(P{R}_{2}H)}^{?}$ (M = Fe, Mo, or W; L = CO or NO; R = Ph or Me). The monoanionic complexes can be deprotonated with n-BuLi at ?78 °C to the corresponding unsymmetric dianions $L_n\stackrel{︹}{M(\mu -P{R}_2)(\mu -L)M}{L}_{n?1}{(P{R}_2)}^{2?}$ (M = Fe; L = CO or NO; R = Ph) or symmetric dianions L_nM(μ-PR₂)₂ML_n^2? (M = Mo or W; L = CO; R = Ph). The unsymmetric dianions isomerize on slight warming to the symmetric dianions, which undergo protonation by CF₃COOH to yield the aforementioned monoanions. Reactions of several members of these three classes of binuclear anions with CF₃COOH, alkylating reagents, 1,1-diiodohydrocarbons and metal diiodo complexes have resulted in the synthesis of new binuclear and trinuclear compounds. Examples include ${(CO)}_3(H)\stackrel{︹}{Fe(\mu -PP{h}_2)Fe}{(CO)}_3(PP{H}_{2}H)$, ${(CO)}_3\stackrel{︹}{Fe(\mu -PP{h}_2)(\mu -C(R)O)Fe}{(CO)}_2(PP{h}_{2}R)$ (R = Me, Et, n-Pr, or i-Pr), ${(CO)}_4\stackrel{︹}{M{(\mu -PP{h}_2)}_{2}M}{(CO)}_3(C(R)Ome)$ (M = Mo or W; R = Me or Ph), ${(CO)}_2({\eta }^3?C_3{H}_5)\stackrel{︹}{Fe(\mu ?PP{h}_2)?F}e{(CO)}_3(PP{h}_2{C}_3{H}_5)$, ${(CO)}_4\stackrel{︹}{M{(\mu ?PP{h}_2)}_{2}M}{(CO)}_3(C(R)Ome)$, ${(NO)}_2\stackrel{︹}{Fe(\mu ?C{H}_2)(\mu ?P{h}_{2}PPP{h}_2)Fe}{(NO)}_2$, and Fe₂Co(η⁵-C₅H₅)(CO)(NO)₄(μ-PPh₂)₂. Synthetic and mechanistic studies on these reactions are presented.