Strategy in Metabolic Regulation
Study on metabolism of small molecules is called as intermediary metabolism in biochemistry is the main subject in a classical biochemical sense.
Living Cell Require a Steady Supply of Starting Materials and Energy- a view from material conservation in living cells.
Living cells can be defined as a steady state system of materials and energy.
Factors : energyand materials(starting materials for synthesis of macromolecules)
Regulation of these factors in a steady state is the living phenomenon
Energy is classified into two categories; phosphorylation status(ATP-ADP ratio) and oxidation status. Two states are closely related and regulated relatively independently.
Organisms Differ in Sources of Energy, Reducing Power, and Starting Materials for Biosynthesis
The following factors are very important in consideration of life style
How energy is supplied?
How energy is stored?
How materials are supplied?
Biochemical Reactions Are Organized into Sequences or Pathways
The intermediates in a biological sequence(eg. Glycolysis) are often exclusive(involved only in glycolysis so that never occur in other metabolic pathway).
These are often reactive or cytotoxic when excess amount is produced.
Sequentially Regulated Enzymes Are Frequently Clustered
Physically and functionally clustered in a cell (metabolon)
may
- accelerate formation of product.
- increase efficacy of intermediate utilization.
- make the regulation effective.
Three Types
- located in the same cellular compartment (glycolysis, DNA synthesis)
- keep high concentration of enzymes and substrates
- reduce possibility of misusing of another enzymes in other pathways for the substrates in a pathway.[the enzymes in fatty acid catabolism(mitochodria) are separated from the enzymes in fatty acid synthesis(cytosol)]
- making aggregate to form a complex (fatty acid synthesis in E. coli) :
- membrane-mediated accumulation (the enzymes of electron transport and oxidative phosphorylation)
Exaamples
A potential role of the cytoskeleton of Saccharomyces cerevisiae in a functional organization of glycolytic enzymes
Gotz R, Schluter E, Shoham G, Zimmermann FK
Zimmermann FK Tech Univ Darmstadt, Inst Mikrobiol & Genet Schnittspahnstr 10 D-64287 Darmstadt Germany Tech Univ Darmstadt, Inst Mikrobiol & Genet D-64287 Darmstadt Germany Hebrew Univ Jerusalem, Inst Chem IL-91904 Jerusalem Isra
Yeast , V.15 N.15 , 1619-1629 , 19991101
abstract :
Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme. This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme. Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons. Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells. We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein. Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production. However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells. However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift. Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains. These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast.
MODEL OF A QUINARY STRUCTURE BETWEEN KREBS TCA CYCLE ENZYMES - A MODEL FOR THE METABOLON
Velot C, Mixon MB, Teige M, Srere PA
Srere PA UNIV TEXAS SW MED CTR DEPT BIOCHEM 4500 S LANCASTER RD DALLAS, TX 75216 USA UNIV TEXAS SW MED CTR DEPT BIOCHEM DALLAS, TX 75216 USA UNIV TEXAS SW MED CTR DEPT VET AFFAIRS MED CTR RES SERV DALLAS, TX 75216 USA
Biochemistry , V.36 N.47 , , 19971125
Abstract :
The enzymes which are responsible for catalyzing sequential reactions in several metabolic pathways have been proposed to be highly organized in supramolecular complexes termed metabolons. However, the in situ existence of these weak complexes is difficult to demonstrate because many of them are dissociated during isolation due to dilution effects. Consequently, the metabolon concept is subject to controversy. A model system consisting of genetically prepared bienzymatic fusion proteins has been used to immobilize sequential metabolic enzymes in close proximity and to demonstrate possible kinetic advantages of metabolons. These experiments use the sequential Krebs TCA cycle enzymes from yeast mitochondrial malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (AGO). Using the porcine high-definition structures of these three enzymes, we have performed computer-modeling studies in order to understand how the molecules may interact. Among the thousands of docking orientations we have tried, one was found to respond to the structural and experimental constraints from the results obtained with the yeast fusion proteins. Interestingly, this quinary structure model shows substantial interacting surface areas with spatial and electrostatic complementarities which make the complex thermodynamically stable, This structure also contains an unbroken electrostatically favorable channel connecting the active sites of ACO and CS, as well as the one previously reported between CS and MDH active sites. Charged amino acids which could be involved in interactions stabilizing the complex have been identified. This model will be used as the basis for further experimental work on the structure of the Krebs TCA cycle metabolon.
EVIDENCE FOR ELECTROSTATIC CHANNELING IN A FUSION PROTEIN OF MALATE DEHYDROGENASE AND CITRATE SYNTHASE
Elcock AH, Macammon JA
Elcock AH UNIV CALIF SAN DIEGO DEPT CHEM & BIOCHEM LA JOLLA, CA 92093 USA UNIV CALIF SAN DIEGO DEPT PHARMACOL LA JOLLA, CA 92093 USA
Biochemistry , V.35 N.39 , , 19961001
Abstract :
Brownian dynamics simulations were performed to investigate a possible role or electrostatic channeling in transferring substrate between two of the enzymes of the citric acid cycle, The diffusion of oxaloacetate from one of the active sites of malate dehydrogenase (MDH) to the active sites of citrate synthase (CS) was simulated in the presence and absence of electrostatic forces using a modeled structure for a MDH-CS fusion protein. In the absence of electrostatic forces, fewer than 1% of substrate molecules leaving the MDH active site are transferred to CS, When electrostatic forces are present at zero ionic strength however, around 45% of substrate molecules are successfully channeled, As expected for an electrostatic mechanism of transfer, increasing the ionic strength in the simulations reduces the calculated transfer efficiency. Even at 150 mM however, the inclusion of electrostatic forces results in an increase in transfer efficiency of more than 1 order of magnitude. The simulations therefore provide evidence for the involvement of electrostatic channeling in guiding substrate transfer between two of the enzymes of the citric acid cycle, Similar effects may operate between other members of the citric acid metabolon.
Pathways Show Functional Coupling
Pathway는 연관된 기능끼리 간단하게 도식화될 수 있다.
catabolism : degradative metabolism
electron and carbon skeleton-liberation from foods :
The electrons can be used to form ATP and the reducing power(NADPH)
anabolism : biosynthesis
utilization of energy, reducing power and carbon skeleton
이 두 과정은 서로 밀접한 연관성을 가지고 동시에 조절되고 있다.
따라서 ATP, NADPH와 small carbon molecule은 이 두 과정의 매개체이며 coupling 을 담담하고 있다.
다른 한 측면은 precursor and end-product realtionship(fig. 12.5)
three types in metabolic compounds
1. central metabolic compounds : exclusive to other pathways
2. 다른 과정에도 사용될 수 있는 중간체들
3. compounds in other pathways
The ATP-ADP System Mediates Conversions in Both Directions
Analysis of metabolism
Conversion : 한 화합물이 다른 화합물로 바뀌는 과정을 의미(방향이 지정되어 있음)
Sequence : conversion이 일어나기 위한 reaction set
어떤 Conversion이 catabolic하면 그 역반응(다른 방향으로의 conversion)은 반드시 anabolic하다. 이 두 과정은 많은 경우 같은 세포 내에서 일어나며 하지만 다른 시간대에 상황에 맞게 벌어진다.
정반대의 conversion이 동시에 일어날 경우 각 conversion은 thermodynamically favorable하게 design된다. 따라서 경우에 따라 열역학적으로 unfavorable 할 경우 ATP가 투입되어 진행시킨다. 이 경우 anabolic conversion에 사용되는 ATP의 양이 catabolic conversion에 사용되는 ATP의 양보다 대개 많다.(Fig. 12.7)
Conversions Are Kinetically Regulated
양방향의 conversion이 동시에 active할 경우 그림 12.7에서 보는 바와 같이 두 conversion은 cycle을 만들게 되며 net-result는 ATP의 소모이다.(futile cycle) 이러한 과정은 에너지의 소모이며 이를 방지하기 위해 각 conversion은 대개 다른 시간대에, 상황에 맡게 진행되는데 따라서 이는 진짜 cycle은 아니다. 이런 의미에서 이를 pseudocycle이라 한다.
Pathways Are Regulated by Controlling Amounts and Activities of Enzymes
Regulation of enzyme activity :
- noncovalent interaction with small regulatory molecules
- reversible covalent modification (phosphorylation and adenylation)
Key Points in Enzymatic Regulation
Regulation은 효소반응의 일부분에서만 이루어진다.
Fig 12.8 Three Types of Enzymatic Regulation
(b)의 특징
한쪽 과정을 억제할 경우 다른 과정은 자동적으로 증가된다.
Cross activation의 경우
Fig. 26.16 GMP 합성의 증가는 AMP 합성의 증가를 유발하여 균형된 생성에 기여한다.
Conversion의 driving force는 sequence의 모든 반응이 열역학적으로 favorable 할 필요는 없고 일부 반응이 ATP 등을 소모하여 전체 sequence가 exergonic 하게 된다.
Behavior of Regulatory Enzymes
가장 전형적인 모습은 cooperative 하다.
Regulatory response를 증가하기 위한 기전임.
hyperbolic dependency
sigmoidal dependency : physiological level의 기질 농도에서 turning point가 형성되어 있는 경우가 많음.
Regulation and Energy Status in Cell
Def. of energy charge : 보유할 수 있는 adenine 관련 phosphate bond 중 hydrolysis가 일어날 수 있는 bond의 비율
energy charge에 따라 catabolism과 anabolism의 비율이 조절됨.
Strategies for Pathway Analysis
analysis of single step pathways :
Substrate를 효소반응을 시킨 후 product를 분석하고 이 반응을 보내는 효소를 추적하여 분리함. 다른 cofactor가 있을 경우 상황이 복잡해짐.
analysis of multiple pathways :
Complementation analysis
- generation of mutants
- Pairwise mating to identify complementation group : 같은 group에 속하는 mutant는 상호 rescue하지 못한다.
- Complementation group의 수는 matabolic sequence에 관여된 gene의 개수와 일치한다.
Biochemical Analysis
- Mutant의 extract를 분석하여 축적된 화합물을 확인하는 작업. 축적된 화합물은 metabolic sequence 상의 중간체일 가능성이 많음.
- 확인된 화합물을 중심으로 이의 생성과정을 담당하고 있는 효소를 분리 확인함.
Radioisotope Tracing
Radioisotope를 가진 화합물을 사용하여 이로부터 생성된 화합물을 확인하여 화합물들 사이의 conversion을 확인하는 작업. 이는 화합물의 conversion을 담당하는 효소를 분리하는데 기초작업임.
Inhibitor의 사용
Mutant의 제공이 용이하지 않을 경우 주로 사용되는 방법. Mutant와 동일한 이유로 사용됨.
Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
The Synthesis and Breakdown of Sugars
Anabolism and Catabolism : biosynthetic pathway and bio-degrading pathway
At a given time, only one of these two processes is active and the other is inactivated.
다른 고분자간의 상호작용 상에서는 모두 한 pathway 상에 있지는 않다. 예로 glycolysis의 진행(a catabolism)은 fatty acid synthesis(an anabolism)와 연계될 수도 있다.
Anabolism과 catabolism의 핵심에는 특정 분자들이 관여하고 있다: key intermediates in biological metabolism. 예로 acetate의 acetylCoA 등 여러 형태는 다음과 같이 중간체적인 역할을 한다.
Overview of Glycolysis
Glycolysis : conversion from glucose or other hexoses to pyruvate (Embden-Meyerhof-Parnas Pathway) and then to lactate, alcohol or CO2
Ubiquitous : occurs in almost every cell
Amphibolic :
catabolic to provide energy or anabolic to provide carbon for other molecules
Not compartmentalized : generally in cytosol
It can be active regardless the presence of oxygen.(aerobic and inaerobic)
구성 : Two Views of Glycolysis
A)metabolic pools : carbon source로서의 관점. three metabolic pools and glycerate-1,3-bisphosphate
metabolic pool : 중간체가 상호 빠른 속도로 치환될 수 있어 상호간 농도는 거의 그 반응의 평형치에 접근한 값을 가진다. 이 경우 상대치는 어떤 경우에도 일정하게 유지된다.
Three pools : hexose phosphate pool, triose phosphate pool I(glyceraldehyde pool), and triose phosphate pool II(glycerate pool)
B)Energy-consuming and paying-off phases :
A)Metabolic Pool : A metabolic cluster which does not require ATP or NADH-driven reaction. Interconversion of the intermediates in the same metabolic pool is relatively rapid. Usually, an entry reaction to a metabolic pool is important in regulation.
The First Metabolic Pool : Three hexose Phosphates
Entry Reactions to Hexose Phosphate Pool :
a) glucose-1-phosphate : the first product in the utilization of polysaccharides
b) glucose-6-phosphate : an intermediate of glycolysis from glucose
c) fructose-6-phosphate : a hexose phosphate from gluconeogenesis and photosynthesis.
The roles of the first metabolic pool : This metabolic pool provides starting materials for some major metabolism :
a) glucose-1-phosphate : polysaccharide synthesis
b) glucose-6-phosphate : pentose phosphate pathway
c) fructose-6-phosphate : glycolysis
Notes for the first pool :
1) Glycogen Phosphorylase : inside cells
(1) glycogen (n-mer) + ATP glycogen ((n-1)-mer) + glucose-1-phosphate.
(2) A similar reaction outside the cells: glucosidases in the intestine to yield glucose finally.
The products from (1) and (2) are different: glucose-1-phosphate and glucose
The product of (1), glucose-1-phosphate, can not pass through the cell membrane. So, they will be utilized for other metabolisms in the cell, such as glycolysis
The product of (2), glucose, can diffuse into the cell and will be phosphorylated to form glucose-6-phosphate.
2) Hexokinase and glucokinase : carry out the same reaction.
hexokinase : its Km for glucose is 10 to 20 M
glucokinase : its Km for glucose is approximately 10 mM and it exists in liver(isozyme type IV).
In liver, only when blood glucose level is very high, glucokinase will form glycogen via the hexose phosphate pool.
3) Regulation of the pool : hormonal regulation via cAMP-dependent phosphorylation.
The Entry to the Second Pool : Formation of Fructose-1,6-bisphosphate
The second pool : From fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glycaldehyde-3-phospahte
The entry to the second pool requires utilization of ATP and is regulated by energy charge and hormones.
After formation of fructose-1,6-bisphosphate, the molecules are utilized only for glycolysis.
Glycerate -1,3-phosphate : glycerate bisphosphate mutase에 의해 glycerate-2,3-bisphosphate로 변한다. 이는 적혈구의 조직으로의 산소운반을 촉진시키는데 관여하고 있다. 따라서 glycolysis가 활발하게 진행될 때에는 이의 중간체인 glycerate-1,3-phosphate의 농도, 따라서 glycerate-2,3-bisphosphate의 농도가 증가하여 세포로의 산소운반을 증가시켜 차후의 oxidative phosphorylation에 준비한다.
The entry to the third pool via formation of glycerate-1,3-bisphosphate
the third pool : glycerate-3-phosphate, glycerate-2-phosphate and phosphoenol pyruvate
the entry to the third pool is closely related with fate of pyruvate. If NAD+ is limited(NADH concentration is high), the entry to the third pool will be limited.
The Fate of Pyruvate :
1) TCA cycle : aerobic process
2) Lactate formation by lactate dehydrogenase : anaerobic process : muscle exertion
3) Alcohol Formation : anaerobic process of yeast : the survival strategy of yeast.
B)Energy-consuming and paying-off phases :
Regulation of Glycolysis : a general consideration
각기의 반응속도는 효소반응이 느리고 기질의 양이 충분한 경우 효소의 활성에 전적으로 의존한다 : enzyme-limited step.
반대로 효소의 활성이 충분하지만 기질의 양이 충분하지 않은 경우 기질의 증감에 따라 속도가 달라진다 : substrate-limited step
전체 pathway는 이러한 특성의 결합으로 이루어져 있으며 이 pathway의 metabolic flux는 그 중 소수의 반응에 의해 좌우된다. : 수학적인 model도 가능.
Metabolic Channels
다른 carbohydrate의 glycolysis
Gluconeogenesis
hexoses and storage polysaccharide formation from lactate, pyruvate and amino acids
glycolysis and gluconeogenesis occur in the same cellular location, cytosol : usually, the oppositely directed biochemical sequences occur in separated location. In this case, they happen at the same site, but by the different enzymes.
Compared to glycolysis, gluconeogenesis is energy-consuming process
The gluconeogenetic enzymes different from those in glycolysis are involved in the interconversion between the metabolic block.
The Pentose Phosphate Pathway
Definition : Formation of pentoses and carbon dioxide from hexoses.
The pentoses are utilized for nucleic acids and other sugars from three to seven carbons.
Use of the pentose pathway : considerably variable
1) muscles : lack of the pentose pathway
2) red blood cells : very active to provide NADPH which is the main reducing power for maintenance of hemoglobin as its reduced form.
Relationship between hexose and pentose
1) Production of Ribulose-5-phosphate from Glucose-6-phosphate: the first step in pentose phosphate pathway
2) Production of Sedoheptulose-7-phosphate from Fructose-6-phosphate
Entry to the nucleic acid synthesis form pentose pathway
Formation of Ribose-5-phosphate from Ribulose-5-phosphate by Ribosephosphate isomerase
The relationship between nucleic acids and glycolysis is not clear.