Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology最新文献

Human α-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe⁸ to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat β-chymase 1, are much less selective, destroying AT by cleaving at Tyr⁴. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys⁴⁰ or Arg¹⁴³ accounts for the human enzyme’s marked preference for Phe⁸ over Tyr⁴. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys⁴⁰ or Arg¹⁴³ to neutral residues. Lys⁴⁰ was exchanged for alanine, the residue found in canine α- and rat β-chymase 1, the latter being dramatically less selective for hydrolysis at Phe⁸. Arg¹⁴³ was exchanged for glutamine found in rat β-chymase 1. The Lys⁴⁰Ala mutant is a dog-like enzyme retaining strong preference for Phe⁸ but with Tyr⁴ hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg¹⁴³Gln mutant, contrary to prediction, remains highly Phe⁸-selective. Therefore, Lys⁴⁰, but not Arg¹⁴³, contributes to human chymase’s remarkable preference for AT II generation over destruction.

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-β-D-glucan was not observed. Cellobiose (G₂) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G₂; inhibition constant values (K_i) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-β-cellobioside as substrate), while a K_i of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-β-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of β-cellobioside and β-cellotrioside (MeUmbG_n). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.

Crystals of Limulus hemocyanin subunits IIIa, IIIb and IV are suitable for X-ray diffraction analysis. The three-dimensional structure of subunit IV is determined by molecular replacement and non-crystallographic symmetry averaging methods. A tentative model of subunit IIIa is obtained from a partial data set. Both structures, similar to subunit II, could provide primary structure segments suitable for oligonucleotide probe synthesis.

Kynurenine formamidase (KFase) (EC 3.5.1.9) hydrolyzes N-formyl-l-kynurenine, an obligatory step in the conversion of tryptophan to nicotinic acid. Low KFase activity in chicken embryos, from inhibition by organophosphorus insecticides and their metabolites such as diazoxon, leads to marked developmental abnormalities. While KFase was purportedly isolated previously, the structure and residues important for catalysis and inhibition were not established. KFase was isolated here from mouse liver cytosol by (NH₄)₂SO₄ precipitation and three FPLC steps (resulting in 221-fold increase in specific activity for N-formyl-l-kynurenine hydrolysis) followed by conversion to [³H]diethylphosphoryl-KFase and finally isolation by C4 reverse-phase high-performance liquid chromatography. Determination of tryptic fragment amino acid sequences and cDNA cloning produced a new 305-amino-acid protein sequence. Although an amidase by function, the primary structure of KFase lacks the amidase signature sequence and is more similar to esterases and lipases. Sequence profile analysis indicates KFase is related to the esterase/lipase/thioesterase family containing the conserved active-site serine sequence GXSXG. The α/β-hydrolase fold is suggested for KFase by its primary sequence and predicted secondary conformation. A three-dimensional model based on the structures of homologous carboxylesterase EST2 and brefeldin A esterase implicates Ser162, Asp247 and His279 as the active site triad.

Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle. The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry. Thermal transitions of the EF-Tu·GDP and EF-Tu·guanosine-5′-[β,γ-imido]triphosphate have a cooperative two-state character. Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu. Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein. Despite the fact that strong interactions between elongation factors Tu and Ts from T. thermophilus at 20°C exist, the thermal transition of neither protein in the complex was significantly affected.

We investigated the effects of guanidine hydrochloride (GuHCl) and high pressure on the conformational flexibility of the active site of sweet potato β-amylase by monitoring the sulfhydryl reaction and the enzymatic activity. The reactivity of Cys345 at the active site, one of six inert half cystine residues of this enzyme, was enhanced by GuHCl at concentrations below 0.5 M. A GuHCl-induced change of the active site was also observed through an intensity change in the near-UV circular dichroism (CD) spectrum. On the other hand, the native conformation of sweet potato β-amylase observed through fluorescence polarization, far-UV CD spectrum and intrinsic fluorescence was not influenced by GuHCl at concentrations below 0.5 M. Therefore, Cys345 reaction caused by GuHCl was due to an alteration of the local conformation of the active site. GuHCl-induced reaction of Cys345, located in the vicinity of subsites 3 and 4, is attributed to enhanced subsite flexibility, which is responsible for substrate slipping in a single-chain attack mechanism. Due to the flexible conformation, the local region of the subsite is more susceptible to GuHCl perturbation than the molecule overall. The enzymatic activity of sweet potato β-amylase was reversibly inhibited by GuHCl at concentrations below 0.5 M, and kinetic analysis of the enzymatic mechanism showed that GuHCl decreases the k_cat value. High pressure below 400 MPa also inactivated sweet potato β-amylase with an increase in Cys345 reactivity. These findings indicated that excessively enhanced subsite flexibility reduced the enzymatic activity of sweet potato β-amylase.

Prourokinase-induced plasminogen activation is complex and involves three distinct reactions: (1) plasminogen activation by the intrinsic activity of prourokinase; (2) prourokinase activation by plasmin; (3) plasminogen activation by urokinase. To further understand some of the mechanisms involved, the effects of epsilon-aminocaproic acid (EACA), a lysine analogue, on these reactions were studied. At a low range of concentrations (10–50 μM), EACA significantly inhibited prourokinase-induced (Glu-/Lys-) plasminogen activation, prourokinase activation by Lys-plasmin, and (Glu-/Lys-) plasminogen activation by urokinase. However, no inhibition of plasminogen activation by Ala¹⁵⁸-prourokinase (a plasmin-resistant mutant) occurred. Therefore, the overall inhibition of EACA on prourokinase-induced plasminogen activation was mainly due to inhibition of reactions 2 and 3, by blocking the high-affinity lysine binding interaction between plasmin and prourokinase, as well as between plasminogen and urokinase. These findings were consistent with kinetic studies which suggested that binding of kringle 1–4 of plasmin to the N-terminal region of prourokinase significantly promotes prourokinase activation, and that binding of kringle 1–4 of plasminogen to the C-terminal lysine¹⁵⁸ of urokinase significantly promotes plasminogen activation. In conclusion, EACA was found to inhibit, rather than promote, prourokinase-induced plasminogen activation due to its blocking of the high-affinity lysine binding sites on plasmin(ogen).

Electrochemical biosensors have found wide application in food and clinical areas, as well as in environmental field. A large number of articles focused on horseradish peroxidase (HRP)-based biosensors have been published in the last decade, due to the capability of HRP to quantitatively detect the presence of hydrogen peroxide produced in a reaction. At present a large body of multi-enzymatic amperometric biosensors are realized by entrapping HRP together with other enzymes into a polymeric matrix; these systems represent a promising example of simple, low-cost electrochemical tools for the analysis of bioanalytes in solution, such as glucose, choline and cholesterol. Due to the fact that polymers used for HRP entrapping are soluble in organic solvents and that many solvents are strong denaturants of aquo-soluble proteins, in this paper we investigate (in particular, by circular dichroism and electron paramagnetic spectroscopies) the effect of dimethyl sulfoxide, one of the organic solvents employed for polymer solubilization, on the structure and the functionality of HRP, in order to determine the effect induced by the solvent concentration on the structure and activity of the hemoprotein. This is relevant for basic and applied biochemistry, HRP being largely employed in bioinorganic chemistry and sensor area.

Two anti-inflammatory triterpenoids, tormentic acid (TA) and euscaphic acid (EA), were found from the plant Rubus sieboldii. These triterpenoids showed an inhibitory effect against enzymes involved in replication, such as calf DNA polymerase α (pol α) and rat DNA polymerase β (pol β). The IC₅₀ values of TA and EA were 37 and 61 μM for pol α and 46 and 108 μM for pol β, respectively. However, TA and EA did not influence the activities of plant DNA polymerases, DNA primase, human immunodeficiency virus type-1 reverse transcriptase, terminal deoxynucleotidyl transferase, any of the prokaryotic DNA polymerases or DNA and RNA metabolic enzymes tested. TA and EA could prevent the growth of BALL-1 cancer cells, and the LD₅₀ values were 11 and 48 μM, respectively. The cells were halted at G₁ phase in the cell cycle. The mode of action of the triterpenoids against anti-inflammatory activity and their relationships to the DNA polymerase inhibitory activity and cell growth inhibition were discussed.