Human testis angiotensin-converting enzyme: crystal structure of a glycosylation mutant and investigation of a putative hinge-mechanism by normal mode analysis
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Date
2004
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University of the Western Cape
Abstract
Human angiotensin-converting enzyme (ACE) is a key enzyme in the
regulation of blood pressure via the renin-angiotensin and kallikrein-kinin
systems. A number of orally active drugs have been developed over the
years that target somatic ACE, for the treatment of hypertension, myocardial
infarction and congestive heart failure. Protein structural information about
ACE is an important key for the understanding of the mechanism and
substrate-specificity of the enzyme. However, this information has only
begun to be elucidated in the past year, with the solution of crystal structures
of human testis ACE (tACE), and homologues Drosophila AnCE and
human ACE2. tACE is identical to the C-terminal domain of somatic ACE,
which consists of two homologous domains, each having a slightly different
substrate-specificity.
This thesis describes the purification, crystallisation and X-ray crystal
structure-determination of a glycosylation-deficient mutant of tACE, tACEG1,3,
to 2.9 Å. The structure of tACE-G1,3 aligns closely with that of
native tACE, indicating that the mutations did not alter the conformation.
The ability to achieve minimal glycosylation of tACE for crystallisation
purposes via mutation, rather than using expensive glycosidase inhibitors,
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should prove advantageous for further structural studies, such as the study of
the binding of novel inhibitors.
In all of the tACE structures thus far observed, the active site is closed off
from the external medium in a deep cleft, so that it is unclear how a large
substrate molecule could gain access. However, a hinge motion that opens
this cleft has been observed in the structures of ACE2. Temperature factor
and sequence comparison between tACE, tACE-G1,3, AnCE and ACE2
suggests the functional conservation of three flexible loop regions, as well as
the sequence conservation of three constrained regions, involved in the
hinge. Normal mode analysis reveals the intrinsic flexibility of tACE, and
further suggests that a putative open form of tACE would behave similarly
to the open form of ACE2. Based on these indications, a conservation of the
ACE2 hinge-bending mechanism is proposed.
Temperature factor analysis also reveals that subdomain II, containing
bound chloride ions, is more structurally rigid than subdomain I, in all
structures considered.
Based on these results, lines of investigation are suggested that should yield
insight into the mechanisms of action of ACE and its association with
various substrates and inhibitors, ideally aiding in the development of novel
drugs for the treatment of cardiac disease.
Description
Magister Scientiae - MSc
Keywords
Angiotensin converting enzyme, Glycosides