Biochemistry

1. Biochemistry

2. Enzyme Regulation Allosteric regulationCovalent Modifications

3. Aspartate transcarbamoylase ATCase • Organization of ATCase • catalytic unit: 6 subunits organized into 2 trimers • regulatory unit: 6 subunits organized into 3 dimers

4. ATCase in metabolic pathway

5. ATCase’s kinetics (a) Rate of ATCase catalysis vs substrate conc.

6. ATCase’s kinetics (b) ATCase catalysis in presence of CTP; ATP

7. ATCase’s allosteric effectors

8. Why ? • The key to allosteric behavior is the existence of multiple forms for the 4º structure of the enzyme. • Allosteric effector modifies the 4º structure of an allosteric enzyme.

9. Allosteric enzymes • Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it • these substances change the enzyme’s activity by altering the conformation(s) of its 4° structure • Homoallostery: Binding of one substrate favors binding of additional substrates. • Heteroallostery :The kinetics of the enzyme can be controlled by any other substance that, in binding to the protein.

10. Allosteric Effectors • Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an • Allosteric Activators(positive effectors)increase substrate binding and/or the rate of the chemical step (kcat). • Allosteric inhibitors(negative effectors) reduce substrate binding and/or the rate of the chemical step.

11. Allosteric Effects • homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase • heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP • Allosteric Activation • Allosteric Inhibition

12. homotropic effects

13. homotropic effects

14. homotropic effects

15. Heteroallostery

16. The Concerted Model(WMC Model) • Wyman, Monod, and Changeux – 1965 WMC Model explains the sigmoidal effects of Heteroallostery • The enzyme has two conformations • R (relaxed): binds substrate tightly; the active form • T (tight or taut): binds substrate less tightly; the inactive form • in the absence of substrate, most enzyme molecules are in the T (inactive) form • the presence of substrate shifts the equilibrium from the T (inactive) form to the R (active) form

17. Concerted Model • in changing from T to R and vice versa, all subunits change conformation simultaneously; all changes are concerted

18. Concerted Model • an allosteric activator (A) binds to and stabilizes the R (active) form • an allosteric inhibitor (I) binds to and stabilizes the T (inactive) form Figure: Effect of binding activators and inhibitors

20. Sequential Model • Koshland - 1966 • the binding of substrate induces a conformational change from the T form to the R form • the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding

21. Sequential Model Figure (a) Sequential model for cooperative binding of substrate to an allosteric enzyme

22. Sequential Model Figure (b) Allosteric activation and inhibition also occur by the induced-fit mechanism

23. Two types of allosteric enzymes • For an allosteric enzyme, the substrate concentration at one-half Vmax is called the K0.5 • K system: an enzyme for which an inhibitor or activators alters K0.5 • V system: an enzyme for which an inhibitor or activator alters Vmax but not K0.5

24. 例天冬氨酸转氨甲酰酶（ATCase）是一种别构酶，它的活性部位与别构效应物结合部位分别位于不同的亚基上。请问它是如何实现别构调节的？有可能设想别构酶上这两种部位存在于同一亚基上吗？为什么？例天冬氨酸转氨甲酰酶（ATCase）是一种别构酶，它的活性部位与别构效应物结合部位分别位于不同的亚基上。请问它是如何实现别构调节的？有可能设想别构酶上这两种部位存在于同一亚基上吗？为什么？ • 回答：用双底物类似物的结合实验和ｘ－射线晶体结构研究揭示ATCase的别构作用是通过四级结构的变化来实现的：（1）在别构（激活）转变中，调节亚基和催化亚基都经历了大的构像变化，结果催化亚基的催化链彼此靠近形成了最优化的活性部位。（2）ATCase的别构效应是在相当大的立体空间范围内起着调节活性的作用。底物的协同结合和ＣＴＰ的反馈抑制是通过长距离而传递的。通过肽链之间的各个表面的相互作用，信息从一个催化亚基的活性部位传递到其他催化亚基的活性部位（详见王镜岩等主编：生物化学（3ed）pp413-415）。因此，别构效应需要蛋白质提供足够的相对独立而又相互作用的界面和长的调节距离。显然单肽链很难满足这两个条件。尽管某些分子量较大的单肽链可以形成几个结构域，使分子内部出现不同的界面，但是可供调节的距离太短。

25. Allosteric switch

26. Covalent Modifications • There are many enzymes in cells which modify other enzymes: • phosphorylation (by protein kinases, PK) or dephosphorylation(by phosphoprotein phosphatase, PP) of various amino acid side chains (e.g., serine, threonine, tyrosine, and histidine). • proteolytic cleavage(by proteases), e.g. activation of zymogens

27. Kinase cascade • A enzymatic cascade that leads to activation or inactivation of the last enzyme in the cascade by sequential phosphorylation. • Kinase cascade is mediated by hormone-receptor complex • Synthesis and degradation of Glycogen: A hormone-activated kinase cascade leads activation of glycogen synthase(GS) or glycogen phosphorylase (GP)

28. glycogen phosphorylase (GP glycogen synthesase(gs)

29. 肾上腺素的作用机理和骨骼肌细胞中糖原的分解代谢肾上腺素的作用机理和骨骼肌细胞中糖原的分解代谢

30. phosphoprotein phosphatase glycogen phosphorylase kinase (GPK) glycogen phosphorylase glycogen synthase

33. Reproduction of yeast

35. Overview of five MAP kinase pathways in S. cerevisiae

36. Kinase cascade in the mating pathway in S. cerevisiae

37. Structures of MAP kinase Inactive form active form