Non‐steady‐state enzyme reaction dynamics

Kim Kyungwoo,Song Sanggeun,Sung Jaeyoung 대한화학회 2022 Bulletin of the Korean Chemical Society Vol.43 No.3

Michaelis–Menten (MM) enzyme kinetics is and has been widely used since early 20th century. However, the conventional enzyme kinetics is not accurate when the substrate is not in excess or when the steady-state approximation does not hold. The steady-state approximation worsens as the enzyme concentration exceeds its substrate concentration, which is the case in many biological processes. Here, to overcome this issue, we present a novel, quasi-exact solution of the enzyme kinetic equations, which provides the time profiles of the substrate, enzyme-substrate complex, and product concentrations. Our theory provides more accurate results than the previously reported theories for all parameter spaces investigated and yields a new relationship of the enzyme reaction time or the enzyme reaction rate to the substrate and enzyme concentrations. We demonstrate our results for the catalytic reaction of β-galactosidase.

Dynamic Kinetic Resolutions and Asymmetric Transformations by Enzyme-Metal Combo Catalysis

김만주,안양수,박재욱 대한화학회 2005 Bulletin of the Korean Chemical Society Vol.26 No.4

Enzyme-metal combo catalysis is described as a useful methodology for the synthesis of optically active compounds. The key point of the method is the use of enzyme and metal in combination as the catalysts for the complete transformation of racemic substrates to single enantiomeric products through dynamic kinetic resolution (DKR). In this approach, enzyme acts as an enantioselective resolving catalyst and metal does as a racemizing catalyst for the efficient DKR. Three kinds of enzyme-metal combinations - lipase-ruthenium, subtilisin-ruthenium, and lipase-palladium ¬have been developed as the catalysts for the DKRs of racemic alcohols, esters, and amines. The scope of the combination catalysts can be extended to the asymmetric transformations of ketones, enol acetates, and ketoximes via the DKRs. In most cases studied, enzyme-metal combo catalysis provided enantiomerically-enriched products in high yields.

Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase

Laye, Victoria J.,Karan, Ram,Kim, Jong-Myoung,Pecher, Wolf T.,DasSarma, Priya,DasSarma, Shiladitya National Academy of Sciences 2017 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.114 No.47

<P><B>Significance</B></P><P>Combining comparative genomics, mutagenesis, kinetic analysis, and molecular modeling provides a powerful way to explore and understand the structure and function of proteins under extreme and potentially astrobiological conditions. Alignment of closely related cold-active and mesophilic β-galactosidase enzymes from halophilic Archaea, followed by mutagenesis and kinetic analysis, demonstrates the importance of specific amino acid residues in temperature-dependent catalytic activity, while molecular modeling provides a structural framework for their mechanism of action. Such an interdisciplinary approach shows how a very small fraction of conserved residues that are divergent from mesophilic homologs are key to enhancing catalytic activity at cold temperatures and underscores the power of combining genomics and genetics with biochemistry and structural biology for understanding polyextremophilic enzyme function.</P><P>The Antarctic microorganism <I>Halorubrum lacusprofundi</I> harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible <I>csp</I>D2 promoter in the genetically tractable Haloarchaeon, <I>Halobacterium</I> sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate <I>o</I>-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (<I>k</I><SUB>cat</SUB>/<I>K</I><SUB>m</SUB>) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both <I>K</I><SUB>m</SUB> and <I>k</I><SUB>cat</SUB> (three substitutions) or either <I>K</I><SUB>m</SUB> or <I>k</I><SUB>cat</SUB> (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.</P>

Dynamic Kinetic Resolutions and Asymmetric Transformations by Enzyme-Metal Combo Catalysis

Kim, Mahn-Joo,Ahn, Yang-Soo,Park, Jai-Wook Korean Chemical Society 2005 Bulletin of the Korean Chemical Society Vol.26 No.4

Enzyme-metal combo catalysis is described as a useful methodology for the synthesis of optically active compounds. The key point of the method is the use of enzyme and metal in combination as the catalysts for the complete transformation of racemic substrates to single enantiomeric products through dynamic kinetic resolution (DKR). In this approach, enzyme acts as an enantioselective resolving catalyst and metal does as a racemizing catalyst for the efficient DKR. Three kinds of enzyme-metal combinations - lipase-ruthenium, subtilisin-ruthenium, and lipase-palladium –have been developed as the catalysts for the DKRs of racemic alcohols, esters, and amines. The scope of the combination catalysts can be extended to the asymmetric transformations of ketones, enol acetates, and ketoximes via the DKRs. In most cases studied, enzyme-metal combo catalysis provided enantiomerically-enriched products in high yields.

Systems-level Modeling with Molecular Resolution Elucidates the Rate-limiting Mechanisms of Cellulose Decomposition by Cellobiohydrolases

Shang, Barry Z.,Chang, Rakwoo,Chu, Jhih-Wei American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.40

<P>Interprotein and enzyme-substrate couplings in interfacial biocatalysis induce spatial correlations beyond the capabilities of classical mass-action principles in modeling reaction kinetics. To understand the impact of spatial constraints on enzyme kinetics, we developed a computational scheme to simulate the reaction network of enzymes with the structures of individual proteins and substrate molecules explicitly resolved in the three-dimensional space. This methodology was applied to elucidate the rate-limiting mechanisms of crystalline cellulose decomposition by cellobiohydrolases. We illustrate that the primary bottlenecks are slow complexation of glucan chains into the enzyme active site and excessive enzyme jamming along the crowded substrate. Jamming could be alleviated by increasing the decomplexation rate constant but at the expense of reduced processivity. We demonstrate that enhancing the apparent reaction rate required a subtle balance between accelerating the complexation driving force and simultaneously avoiding enzyme jamming. Via a spatiotemporal systems analysis, we developed a unified mechanistic framework that delineates the experimental conditions under which different sets of rate-limiting behaviors emerge. We found that optimization of the complexation-exchange kinetics is critical for overcoming the barriers imposed by interfacial confinement and accelerating the apparent rate of enzymatic cellulose decomposition.</P>

A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1

Huon, Thavrak,Jang, Sung-Kuk,Cho, Kyoung-Won,Rakwal, Randeep,Woo, Je-Chang,Kim, Il-Chul,Chi, Seung-Wook,Han, Ok-Soo Korean Chemical Society 2009 Bulletin of the Korean Chemical Society Vol.30 No.4

The maize lipoxgyenase-1 is a non-traditional dual positional specific enzyme and the reaction proceeds via enzyme-initiated catalysis. Bioinformatic analysis indicated that the maize lipoxygenase-1 is structurally more similar to soybean LOX1 than pea LOXN2 in that it has an additional external loop (residues 318-351) in the carboxy-terminal catalytic domain. We analyzed the dependence of product distribution on concentration of linoleic acid and monitored the formation of hydroperoxyoctadecadienoic acid as a function of enzyme concentration. Product distribution was strongly influenced by substrate concentration, such that kinetically-controlled regioisomers were enriched and thermodynamically-controlled regioisomers were depleted at high substrate concentration. Kinetic studies indicated that the formation of hydroperoxyoctadecadienoic acid saturated rapidly in an enzyme concentration-dependent manner, which implied that reactivation by reoxidation of inactive Fe(II) failed to occur. Our results support the previously proposed enzyme-initiated catalytic mechanism of the maize lipoxgyenase-1 and reveals that a substrate molecule serves as a hydrogen atom donor in its enzyme-initiated catalysis.

A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1

Thavrak Huon,Sungkuk Jang,조경원,Randeep Rakwal,우제창,김일철,지승욱,한옥수 대한화학회 2009 Bulletin of the Korean Chemical Society Vol.30 No.4

The maize lipoxgyenase-1 is a non-traditional dual positional specific enzyme and the reaction proceeds via enzyme-initiated catalysis. Bioinformatic analysis indicated that the maize lipoxygenase-1 is structurally more similar to soybean LOX1 than pea LOXN2 in that it has an additional external loop (residues 318-351) in the carboxy-terminal catalytic domain. We analyzed the dependence of product distribution on concentration of linoleic acid and monitored the formation of hydroperoxyoctadecadienoic acid as a function of enzyme concentration. Product distribution was strongly influenced by substrate concentration, such that kinetically-controlled regioisomers were enriched and thermodynamically-controlled regioisomers were depleted at high substrate concentration. Kinetic studies indicated that the formation of hydroperoxyoctadecadienoic acid saturated rapidly in an enzyme concentration-dependent manner, which implied that reactivation by reoxidation of inactive Fe(II) failed to occur. Our results support the previously proposed enzyme-initiated catalytic mechanism of the maize lipoxgyenase-1 and reveals that a substrate molecule serves as a hydrogen atom donor in its enzyme-initiated catalysis.

Saccharification of Foodwastes Using Cellulolytic and Amylolytic Enzymes from Trichoderma harzianum FJ1 and Its Kinetics

Kim Kyoung-Cheol,Kim Si-Wouk,Kim Myong-Jun,Kim Seong-Jun The Korean Society for Biotechnology and Bioengine 2005 Biotechnology and Bioprocess Engineering Vol.10 No.1

The study was targeted to saccharify foodwastes with the cellulolytic and amylolytic enzymes obtained from culture supernatant of Trichoderma harzianum FJ1 and analyze the kinetics of the saccharification in order to enlarge the utilization in industrial application. T. harzianum FJ1 highly produced various cellulolytic (filter paperase 0.9, carboxymethyl cellulase 22.0, ${\beta}$-glucosidase 1.2, Avicelase 0.4, xylanase 30.8, as U/mL-supernatant) and amylolytic (${alpha}$-amylase 5.6, ${\beta}$-amylase 3.1, glucoamylase 2.6, as U/mL-supernatant) enzymes. The $23{\sim}98\;g/L$ of reducing sugars were obtained under various experimental conditions by changing FPase to between $0.2{\sim}0.6\;U/mL$ and foodwastes between $5{\sim}20\%$ (w/v), with fixed conditions at $50^{\circ}C$, pH 5.0, and 100 rpm for 24 h. As the enzymatic hydrolysis of foodwastes were performed in a heterogeneous solid-liquid reaction system, it was significantly influenced by enzyme and substrate concentrations used, where the pH and temperature were fixed at their experimental optima of 5.0 and $50^{\circ}C$, respectively. An empirical model was employed to simplify the kinetics of the saccharification reaction. The reducing sugars concentration (X, g/L) in the saccharification reaction was expressed by a power curve ($X=K{\cdot}t^n$) for the reaction time (t), where the coefficient, K and n. were related to functions of the enzymes concentrations (E) and foodwastes concentrations (S), as follow: $K=10.894{\cdot}Ln(E{\cdot}S^2)-56.768,\;n=0.0608{\cdot}(E/S)^{-0.2130}$. The kinetic developed to analyze the effective saccharification of foodwastes composed of complex organic compounds could adequately explain the cases under various saccharification conditions. The kinetics results would be available for reducing sugars production processes, with the reducing sugars obtained at a lower cost can be used as carbon and energy sources in various fermentation industries.

Cloning and Expression of Escherichia coli Ornithine Transcarbamylase Gene, argI

류기중,유장걸,고영환,김찬식,송성준,오영선,이선주,Riu, Key-Zung,U, Zang-Kual,Ko, Young-Hwan,Kim, Chan-Shik,Song, Sung-Jun,Oh, Young-Seon,Lee, Sun-Joo 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.2

Escherichia coli의 오르니틴 트란스카바밀라제는 오르니틴과 카바밀인산으로부터 시트룰린의 합성을 촉진시키는 효소이다. 이 효소의 기능과 구조와의 상관관계, 반응메카니즘 등 생화학적 연구를 하기 위하여 대량의 효소를 추출할 필요가 있다. 본 연구는 오르니틴 트란스카바밀라제의 대량생산 시스템을 확립하기 위하여 E. coli argI 유전자를 E. coli $DH5{\alpha}$ 세포의 염색체 DNA를 추출한 후에 PCR 방법으로 증폭시켜 얻었다. 증폭된 argI 유전자를 단핵생물 단백질 발현벡터인 pKK223-3에 접합시킨 후, 오르니틴 트란스카바밀라제가 존재하지 않은 E. coli TB2 세포에 클로닝 시켰다. 이 세포로부터 생산된 오르니틴 트란스카바밀라제는 암모늄염에 의한 분할, 열변성, 크로마토그래피 등을 사용하여 순수하게 분리하였다. SDS 단백질 전기영동 결과 약 38 kDa 크기의 효소가 순수하게 얻어졌다. 반응속도론적 실험결과 $K_{cat}$은 $1{\times}10^5m^{-1}$, $K_M$은 오르니틴에 대하여는 0.35 mM, 카바밀인산에 관하여는 0.06 mM이 각각 얻어졌다. 이 결과는 야생형 오르니틴 트란스카바밀라제의 반응속도 인자들과 비슷한 값이다. 본 연구는 이들 결과로부터 오르니틴 트란스카바밀라제의 기능을 하는 E. coli argI 유전자가 클로닝 되었음을 확인하였다. Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of $DH5{\alpha}$, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the s?????? values as those of the wild type enzyme. The $k_{cat}$, of the enzyme was $1.0{\times}10^5min^{-1}$, and $K_ms$ for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.

Kinetic limitations of a bioelectrochemical electrode using carbon nanotube-attached glucose oxidase for biofuel cells

Zhao, Xueyan,Jia, Hongfei,Kim, Jungbae,Wang, Ping Wiley Subscription Services, Inc., A Wiley Company 2009 Biotechnology and bioengineering Vol.104 No.6

<P>Carbon nanotubes (CNTs) have been used for various bioelectrochemical applications, presumably for substantial improvement in performance. However, often only moderate results observed, with many governing factors have been considered and suggested yet without much systematic evaluation and verification. In this study, CNT-supported glucose oxidase (CNT–GOx) was examined in the presence of 1,4-benzoquinone (BQ). The intrinsic Michaelis parameters of the reaction catalyzed by CNT–GOx were found very close to those of native GOx. However, the Nafion entrapment of CNT–GOx for an electrode resulted in a much lower activity due to the limited availability of the embedded enzyme. Interestingly, kinetic studies revealed that the biofuel cell employing such an enzyme electrode only generated a power density equivalent to <40% of the reaction capability of the enzyme on electrode. It appeared to us that factors such as electron and proton transfer resistances can be more overwhelming than the heterogeneous reaction kinetics in limiting the power generation of such biofuel cells. Biotechnol. Bioeng. 2009; 104: 1068–1074. © 2009 Wiley Periodicals, Inc.</P>