Lecture 23 Summary: Introduction to metabolism

The regulatory mechanisms we have been studying by way of examples such as hemoglobin and the blood clotting cascade are rich with physiological and clinical implications and connections. We discuss one such case, the advantage of the heterozygous carrier of the gene at the β locus for HbS, i.e. resistance to malaria, and the molecular and cellular basis of this phenomenon. External link: Malaria and the Red Cell (see ref 2 below).

The proteolytic activation regulatory strategy is capable of using a relatively small number of activated molecules to turn on a great many others, thus amplifying an initial activation response. However, the activation is not readily reversible (as can be the case in phosphorylation) since once activated, the proteolytically processed species remain active unless they are degraded or targeted by specific inhibitors. Examples of the latter are pancreatic trypsin inhibitor and members of a class of serine protease inhibitors called serpins. Two members of the serpin class discussed in the text are α₁-antitrypsin and antithrombin III.

The blood clotting cascade (Ch.10, pp.293-297). The role of Vitamin K in modification of the pro-region of prothrombin.

Activation of enzymes by specific proteolytic cleavage

We finish our consideration of regulatory strategies by learning about a case of activation by proteolytic cleavage that is of obvious physiological importance - the blood clotting cascade. A series of proteolytic activation events, each of which is similar to the activation of the zymogen forms of the pancreatic serine proteases, leads to the formation of blood clots in response to injury or trauma. This blood clotting pathway has become the classic example of a zymogen activation cascade. A key feature of this cascade is the amplification of an initial triggering event into a major physiologic response.

Intrinsic pathway

* Triggered by the "non-physiological surface" of an injury
* Begins with activation of Factor XII (Hageman factor)

Extrinsic pathway

* Trauma triggers activation of Factor VII
* Release of lipoprotein called tissue factor follows

Final common pathway

* Intrinsic and extrinsic pathways converge, both activating Factor X
* The end result is the conversion of fibrinogen to fibrin by thrombin

Thrombin - yet another serine protease

Activated thrombin is responsible for the conversion of fibrinogen to fibrin. It shows a specificity for cleavage of the peptide bond between Arg and Gly, suggesting a similarity to trypsin. Indeed, the B chain of thrombin shows sequence similarity to trypsin, and the X-ray structure of thrombin reveals all the hallmarks of a serine protease: the catalytic triad, oxyanion hole, and specificity pocket. The latter, like trypsin, has an aspartate residue at its bottom that interacts favorably with the Arg at P₁ of the substrate.

Thrombin arises from the processing of an inactive precursor, or zymogen, called prothrombin. Prothrombin is a 582-residue polypeptide whose first 274 residues constitute the large "pro" region. Processing (proteolytic cleavage) by Factor Xa (stimulated by Factor Va) corresponds to cleavage of the Arg274-Thr275 and Arg323-Ile324 peptide bonds. The pro region is released, while the two fragments of the mature polypeptide remain associated and are covalently joined by a disulfide bond.

The pro region of thrombin contains (near the N-terminus) a number of modified Glu residues that contain an extra carboxyl group. These residues, called γ-carboxyglutamate (three-letter abbreviation Gla), act as effective chelators of Ca²⁺ ions. This property of prothrombin is essential for the proper functioning of the clotting cascade. This is because the binding of calcium ions by prothrombin anchors it to phospholipid membranes derived from platelets following injury. This properly localizes prothrombin in proximity to its activating factors.

Learning objectives

• Explain how a homozygous carrier of the sickle-cell gene could have a selective advantage against malaria
• Describe thrombin and its role in the final common pathway of blood clotting.
• Describe the role of vitanin K in the blood clotting process.

Page update in progress, 11-07-09

References

1. Staunton J. Primary Metabolism: A Mechanistic Approach (1978, Oxford University Press)
2. Information Center for Sickle Cell and Thalassemic Disorders. (hosted by Harvard Medical School and Brigham and Women's Hospital)
3. Soriano-Garcia M, Padmanabhan K, de Vos ]AM, Tulinsky A. (1992). The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry 31: 2554-2566.