CH6059 Advanced Physical Chemistry: Hirudin's Structure & Inhibition

This essay provides a detailed analysis of hirudin, a naturally occurring peptide with anticoagulant properties, focusing on its structure, role, and mechanism of action as an enzyme inhibitor. It discusses hirudin's interaction with thrombin, highlighting its ability to inhibit thrombin's procoagulant activity and prevent clot formation. The essay also explores various recombinant hirudin derivatives and their therapeutic applications, particularly in blood coagulation disorders. Furthermore, it delves into the kinetics of enzyme reactions, including the determination of Km and Vmax values, and examines different types of enzyme inhibition, providing a comprehensive overview of hirudin's significance in advanced physical chemistry. Desklib is a great platform to find similar solved assignments.

ADVANCED PHYSICAL CHEMISTRY
By Name
Course
Instructor
Institution
Location
Date

Hirudin
Abstract
It refers to a peptide that occurs naturally in the salivary glands of the blood-sucking leeches.
The examples of such blood leeches include Hirudin medicinalis which is known to be
having blood anticoagulant property. This property is very crucial for the alimentary habit of
the hematophagy shown by the leeches considering that it keeps the blood flowing after the
first operation of the phlebotomy is carried out on the skin of the host by the worm (Sellers
et.al 2014).
Structure
During the times of the great scientists John Berry, there were publications of the paper that
dealing with the coagulation of the blood. In his work, he discovered that the leech secreted a
very powerful anticoagulant and he refers to the anticoagulant as the hirudin. The isolation of
this structure was not done until the year 1950 with the determination of its full structure
being finished on the year 1976.The full length of this structure is made up of roughly 65
amino acids.
The present amino acids are arranged in the form of a solid N-terminal domain possessing
three disulphide bonds and a terminal of C. The C compound is perfectly disordered when the
protein is subjected to the uncompleted solution. The residues of the amino acids normally
form a parallel beta strand of 1-3 to the residues of thrombin 214-217.The ser-195 O gamma
atom present in the site of the catalytic will form the hydrogen bond with the nitrogen atom
of the residue 1. There are several electrostatic interactions between the C-terminal domain
and the anion-binding of the thrombin exosite (Lu et.ai 2013)
The last five residues form a loop that is helical and this results into hydrophobic contacts.
There are several mixtures of the isoforms of the protein in the natural Rudin. The

homogeneous preparation of the hirudin can be an achieved using a technique of the
recombinant.
Figure 1: Structure of hirudin extracted from Yingxin et.al 2014
In the final stages of the coagulation of the blood, the most common activity is the conversion
of the fibrinogen into the fibrin. This is done by an enzyme called serine protease thrombin.
The production of the thrombin is from the prothrombin and this is facilitated by an enzyme
called prothrombinase towards the end of the coagulation stages.
The activity includes the use of the Factor Xa together with the Factor Va as the cofactor
especially in the final stages of the coagulation. A blood clot is formed through linking of the
fibrin by the factor XIII. The XIII is also known as the fibrin stabilizing factor. The primary
inhibitor of the thrombin in the normal circulation of the blood is the antithrombin. The
anticoagulant activity of the hirudin is based on its capability to inhibit the activity of the
thrombin of procoagulation. This is what makes it similar to the antithrombin.
Hirudin is the primary natural inhibitor of the thrombin. It normally binds to and inhibit only
the thrombin that ha been activated. This limits its activities to specifically on the
fibrinogen’s hirudin therefore prevents the formation of the clots and the thrombi. This makes
the activity to be a thrombolytic.

Hirudin has a therapeutic value in the blood coagulation disorders and in the treatment
process of the skin hematomas together with the superficial varicose veins. It can be used
either as an injection or as a cream to be applied. The advantage of the hirudin over other
commonly used anticoagulant and thrombolytics like heparin is that it does not trigger any
interference with the biological operations of the present serum proteins. Besides it can act as
a very complexed thrombin.
Role as an inhibitor
The compounds such as lepirudin and desirudin are commonly known as the recombinant
hirudin derivatives that inhibits directly free and fibrin bound thrombin thereby blocking its
activities. There exist also a group of the derivatives such as Bivalirudin, argatroban among
others that are synthetic. The derivatives of the hirudin and the hirudin itself acts as a bivalent
direct thrombin inhibitor.
The block both the active sites and the exosite. The Bivalirudin binds reversibly to the
compound of the thrombin and this makes its inhibitory effect a transient which consequently
results into the diminished risk of serious bleeding. The univalent direct thrombin inhibitors
that binds only to the active sites of the thrombin includes the Argatroban and gabigatran
etexilate. The approval of the gabigatran was done in the year 2012 as the first new direct oral
anticoagulant treatment option. The use of the direct thrombin is common in the cases where
there is intolerability of the heparin such as the heparin induced.
Consequences of inhibition action.
Recombinant hirudin does not have the sulfidation effects and this leads into a two-fold loss
of potency without putting any change in its specificity of inhibiting thrombin. The
crystallographic structure of X-ray details shows a structure of the hirudin-thrombin that are
coupled in the complex manner (Strassel et.al 2012). The study of the structure indicates that

the amino terminal domain of the hirudin interacts with the active sites of the thrombin’s
catalytic triad residue. This is carried along with the carboxy terminal domain that binds
positively to the charged anion binding exosite (Huang et.al 2012)
When an intravenous dose of hirudin is used, the commonly observed half-life is just
40minutes.This half-life is increased to a round 200 minutes with the injection of the
subcutaneous. The excretion of the hirudin is predominantly done by the kidneys. There is a
prolonged PPT in the dose-dependent manner.
The injection type of subcutaneous of 0.1mg/kg extends the prolong duration almost twice.
However, when given to the volunteers Hirudin does not prolong the bleeding time twice
with the recommended baseline. The studies have shown that the patients with the coronary
arterial and the venous thrombosis have slight decrease in the angioplasty-associated acute
complications with higher risks of bleeding (Guo-Qian, Gang and Zhi-Yong 2012)
It is however, beneficial in the long term. Hirudin has been shown to be slightly better as
compared to heparin in the prevention of the thromboembolism of the venous especially with
the patients that are undergoing the replacement of the hip. This advantage extends even to
the treatment of the established deep vein thrombosis.
QUESTION TWO
(a)The intermediaries present in the reactions include substrate and the enzymes.
(b) X=(E)+(EH)+(EHs)
Y=(Es)+(EHS) +(EHP) +(EH2P)
Z=(EP)+(EHP)+(EHP) the overall expression thus becomes x+y+z=(Es).

(c) For a given reaction aA +bB=C, the rate of reaction was taken as r=K(A)a (B)b in which
k=reaction rate, (A) concentration of A and the concentration of B respectively. Using the
above equation;
X=(E)+(EH)+(EHs)
Y=(Es)+(EHS) +(EHP) +(EH2P)
Z=(EP)+(EHP)+(EHP) the overall expression thus becomes x+y+z=(Es).
1/V0=Km/Vmax(1+[I]/K1) x1/[S]+1/Vmax(1+[I]/K1) and this give the value of
Km/Vmax(1+{I]/K1) = 1132.1 which corresponds to K1=4.1x10-3M
(d)The rate law of the reaction can be obtained using the isolation method of the reactants. In
this particular case the reactant used taken as A+B=Consider Both be in excess in this
reaction and so taking the concentration of b to be constant, the law of expression becomes
V=K(A) K’=K(B)0
(e) When the Concentration of the substrate is taken to be very low. The reaction will shift to
the left and this results into the reduced value of the product. In this particular reaction, other
factors that affect reaction apart from the PH are held constant. This includes the temperature
and pressure (Xu et.al 2013).
QUESTION 3
(a) It states that the rate of the enzymatic reaction in which the substrate S is converted
into the product P depends on the concentration of the enzyme despite the fact that the
enzyme doe not undergo any change.

(b)Determination of the values of Km and Vmax of the enzymes.
According to the graph, the inhibitor A and B, meets at the X-axis thus non-competitive type
of inhibitors. The graphical determination is as shown below
The line with no inhibition crosses the axis of Y at 2.5x105M-1s and this translates to a Vmax
6.12x10-6MS-1. The gradient of the line Km /Vmax=1.88x10-4M (Greinacher2012)
(c) Determination of the type of the inhibition
Since this has been established for the competitive inhibitor;
1/Vo=Km/Vmax(1+[1]/K1) x1/[S]+1/Vmax.

The slope for the competitive inhibitor=Km/Vmax(1+1/K1) =901.6
Substituting for the value of the gradient which 297 and taking int assumptions that [I] is
8.0x10-3Ms, the resultant value of K1 =6.6x10-3M.
Also taking the formula for the calculation of the non-competitive inhibitor; (Yingxin et.al
2013)
1/V0=Km/Vmax(1+[I]/K1) x1/[S]+1/Vmax(1+[I]/K1) and this give the value of
Km/Vmax(1+{I]/K1) = 1132.1 which corresponds to K1=4.1x10-3M

References
Greinacher, A., 2012. Recombinant 14 hirudin for the treatment of heparin-induced
thrombocytopenia. Heparin-induced thrombocytopenia, p.388.
Guo-Qian, Y., Gang, W. and Zhi-Yong, S., 2012. Investigation on the microcirculation effect
of local application of natural hirudin on porcine random skin flap venous congestion. Cell
biochemistry and biophysics, 62(1), pp.141-146.
Huang, Y., Zhang, Y., Wu, Y., Wang, J., Liu, X., Dai, L., Wang, L., Yu, M. and Mo, W.,
2012. Expression, purification, and mass spectrometric analysis of 15N, 13C-labeled RGD-
hirudin, expressed in Pichia pastoris, for NMR studies. PLoS One, 7(8), p. e42207.
Lu, W., Cai, X., Gu, Z., Huang, Y., Xia, B. and Cao, P., 2013. Production and
characterization of hirudin variant-1 by SUMO fusion technology in E. coli. Molecular
biotechnology, 53(1), pp.41-48.
Sellers, D.L., Kim, T.H., Mount, C.W., Pun, S.H. and Horner, P.J., 2014. Poly (lactic-co-
glycolic) acid microspheres encapsulated in Pluronic F-127 prolong hirudin delivery and
improve functional recovery from a demyelination lesion. Biomaterials, 35(31), pp.8895-
8902.
Strassel, C., Eckly, A., Léon, C., Moog, S., Cazenave, J.P., Gachet, C. and Lanza, F., 2012.
Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow
progenitors. Experimental cell research, 318(1), pp.25-32.
Xu, Y., Wu, W., Wang, L., Chintala, M., Plump, A.S., Ogletree, M.L. and Chen, Z., 2013.
Differential profiles of thrombin inhibitors (heparin, hirudin, bivalirudin, and dabigatran) in
the thrombin generation assay and thromboelastography in vitro. Blood Coagulation &
Fibrinolysis, 24(3), pp.332-338.

Yingxin, G., Guoqian, Y., Jiaquan, L. and Han, X., 2013. Effects of natural and recombinant
hirudin on VEGF expression and random skin flap survival in a venous congested rat
model. International surgery, 98(1), pp.82-87.