Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

2011 ◽  
Vol 437 (2) ◽  
pp. 223-230 ◽  
Author(s):  
Han-Woo Kim ◽  
Kazuhiko Ishikawa
Keyword(s):  
Enzyme Reaction ◽  
Intermediate Structure ◽  
Amino Acid Residues ◽  
Pyrococcus Horikoshii ◽  
Substrate Complex ◽  
Substrate Structure ◽  
Enzyme Substrate ◽  
Cellulose Substrates ◽  
Boat Form ◽  
Low Activity

A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65–2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite −1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites −1 and +1. Subsite −2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite −1. A rare enzyme–substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme–substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.

scholarly journals Kinetics of an enzyme reaction in which both the enzyme-substrate complex and the product are unstable or only the product is unstable

1994 ◽  
Vol 303 (2) ◽  
pp. 435-440 ◽  
Author(s):  
C Garrido-del Solo ◽  
F García-Cánovas ◽  
B H Havsteen ◽  
E Valero ◽  
R Varón
Keyword(s):  
Computer Simulation ◽  
Data Analysis ◽  
Kinetic Data ◽  
Enzyme Reaction ◽  
Enzyme Concentration ◽  
Substrate Complex ◽  
Use Of Data ◽  
Enzyme Substrate ◽  
Experimental Errors ◽  
Kinetics Of

A kinetic analysis of the Michaelis-Menten mechanism has been made for the case in which both the enzyme-substrate complex and the product are unstable or only the product is unstable, either spontaneously or as the result of the addition of a reagent. This analysis allows the derivation of equations which under conditions of limiting enzyme concentration relate the concentration of all of the species to the time. A kinetic data analysis is suggested, which leads to the evaluation of the kinetic parameters involved in the reaction. The analysis is based on the equation which describes the formation of products with time and one's experimental progress curves. We demonstrate the method numerically by computer simulation of the reaction with added experimental errors and experimentally by the use of data from the kinetic study of the action of tyrosinase on dopamine.

scholarly journals Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

2003 ◽  
Vol 370 (1) ◽  
pp. 331-337 ◽  
Author(s):  
Paloma SÁNCHEZ-TORRES ◽  
Jaap VISSER ◽  
Jacques A.E. BENEN
Keyword(s):  
Aspergillus Niger ◽  
Conformational Change ◽  
Three Dimensional ◽  
Pectin Lyase ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Fluorescence Studies ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Positively Charged

Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed β-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689] and the modelled enzyme—substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 8762—8769], Asp154, Arg176, Arg236 and Lys239 were mutagenized. Substituting Arg236 with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg176 and Lys239 severely affected catalysis. The Asp154→Arg and Asp154→Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg236, which is sandwiched between Arg176 and Lys239, would initiate the reaction upon enzyme—substrate interaction, through the abstraction of the proton at C5 of the galacturonopyranose ring. The positively charged residues Arg176 and Lys239 are responsible for lowering the pKa of Arg236. Arg176 and Lys239 are maintained in a charged state by interacting with Asp154 or bulk solvent respectively. The deprotonation of the Asp186—Asp221 pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677—689]. Substitution of Asp186 and Asp221 by Asn186 and Asn221 was expected to stabilize the enzyme. However, the Asp186→Asn/Asp221→Asn enzyme appeared less stable than the wild-type enzyme, even at pH6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Asp186—Asp221 pair.

scholarly journals Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase

1999 ◽  
Vol 341 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Satoru NIRASAWA ◽  
Yoshiaki NAKAJIMA ◽  
Zhen-Zhong ZHANG ◽  
Michiteru YOSHIDA ◽  
Kiyoshi HAYASHI
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Amino Acid Residues ◽  
E Coli ◽  
Substrate Complex ◽  
Extracellular Secretion ◽  
Enzyme Substrate ◽  
Aeromonas Caviae ◽  
Km Value

An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.

scholarly journals A graphical method for determining inhibition parameters for partial and complete inhibitors

1987 ◽  
Vol 248 (3) ◽  
pp. 815-820 ◽  
Author(s):  
M Yoshino
Keyword(s):  
Substrate Concentration ◽  
Competitive Inhibition ◽  
Graphical Method ◽  
Enzyme Reaction ◽  
Reaction Rate Constant ◽  
Inhibition Mechanism ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Characteristic Features ◽  
Substrate Inhibitor

A new simple graphical method is described for the determination of inhibition type and kinetic parameters of an enzyme reaction without any replot. The method consists of plotting experimental data as v/(vo-v) versus the reciprocal of the inhibitor concentration at different substrate concentrations, where v and vo represent the velocity in the presence and in the absence of the inhibitor respectively with a given concentration of the substrate. Partial inhibition gives straight lines that converge on the abscissa at a point away from the origin, whereas complete inhibition gives lines that go through the origin. The inhibition constants of enzymes and the reaction rate constant of the enzyme-substrate-inhibitor complex can be calculated from the abscissa and ordinate intercepts of the plot. The relationship between the slope of the plot and the substrate concentration shows characteristic features depending on the inhibition type: for partial competitive inhibition, the straight line converging on the abscissa at-Ks, the dissociation constant of the enzyme-substrate complex; for non-competitive inhibition, a constant slope independent of the substrate concentration; for uncompetitive inhibition, a hyperbola decreasing with the increase in the substrate concentration; for mixed-type inhibition, a hyperbola increasing with the increase in the substrate concentration. The properties of the replot are useful in confirmation of the inhibition mechanism.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

scholarly journals Conformational Analysis of the Enzyme-Substrate Complex in the Dehydrogenation of Sterols by Tetrahymena pyriformis

1971 ◽  
Vol 246 (3) ◽  
pp. 561-568 ◽  
Author(s):  
William R. Nes ◽  
P.A. Govinda Malya ◽  
Frank B. Mallory ◽  
Karen A. Ferguson ◽  
Josephine R. Landrey ◽  
...  
Keyword(s):  
Conformational Analysis ◽  
Tetrahymena Pyriformis ◽  
Substrate Complex ◽  
Enzyme Substrate

scholarly journals N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

2021 ◽  
Vol 49 (5) ◽  
pp. 2684-2699
Author(s):  
Ka-Weng Ieong ◽  
Gabriele Indrisiunaite ◽  
Arjun Prabhakar ◽  
Joseph D Puglisi ◽  
Måns Ehrenberg
Keyword(s):  
Single Molecule ◽  
Stop Codon ◽  
Product Formation ◽  
Release Factors ◽  
Substrate Complex ◽  
Peptidyl Transfer ◽  
Enzyme Substrate ◽  
Peptide Release ◽  
Accuracy Ratio ◽  
Codon Reading

Abstract We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on formation of the enzyme-substrate complex

Biochemistry ◽  
1984 ◽  
Vol 23 (20) ◽  
pp. 4557-4564 ◽  
Author(s):  
Jan L. M. L. Van Rijn ◽  
Jose W. P. Govers-Riemslag ◽  
Robert F. A. Zwaal ◽  
Jan Rosing
Keyword(s):  
Kinetic Studies ◽  
Activation Effect ◽  
Substrate Complex ◽  
Factor Va ◽  
Enzyme Substrate ◽  
Prothrombin Activation
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