Caspase Biochemistry Practical Exam Flashcards
what action do proteolytic enzymes take?
hydrolyse peptide bonds
exothermic
give the four classes of proteolytic enzymes
serine proteases
cysteine proteases
aspartyl proteases
metalloproteases
what are the 6 broad categories of protease function?
nutrition invasion evasion adhesion processing signalling
what sort of specificity must occur for protein binding to take place?
substrate must demonstrate genomertical and physical complementarity to enzyme
for each protease, state which sort of AA it prefers to hydrolyse:
a) serine protease
b) chymotrypsin
c) trypsin
d) elastase
a) position R-(n-1)
b) bulky, aromatic
c) + charged
d) small, non-charged
describe serine proteases
- first protease class identified
- i.e. digestive enzyme trypsin
- active site has 3 residues; Ser, His and Asp (sequentially far apart but structurally close)
- substrate specificity can vary
which type of amino acid residue does chymotrypsin prefer?
an aromatic side chain on reside where α-carbon is part of peptide bond to be cleaved
which type of amino acid residue does trypsin prefer?
positively chrgaed Lys or Arg
name some other common ser proteases
elastase collegenase keratinase trypsin plasmin thrombin
give some examples of cysteine proteases
papain: well-studied plant protease
cathepsins: lysosomal proteases
caspases: cys asp proteases involved in apoptosis
calpains: Ca-activated proteases that cleave IC proteins involved in cell motility and adhesion
what is the catalytic mechanism of a cysteine protease?
cys sulfhydryl group
deprotonation of cys SH by adjacent His residue followed by nucleophilic attack of Cys S on peptide carbonyl carbon
thioester linking new carboxy-terminus to Cys thiol is intermediate of reaction (comparable to acyl-enzyme intermediate of Ser protease)
how is cysteine protease mechanism of papain different to chymotryspin?
similar but extra step required
Cys S not electronegative enough to disrupt O double bond
activated by His = tetrahedron 1 intermediate complex
(see lec slide 7 for more details)
what are the aspartyl proteases?
smallest class of human proteases
include digestive enzymes pepsin, lysosomal cathepsin D
renin (kidney enzyme)
HIV-protease
how do all aspartyl proteases operate?
two Asp residues at active site
two Asps work together as general acid-base catalysts
what structure do most aspartic proteases have?
a tertiary structure consisting of two lobes (N- and C- terminal) with approximate two-fold symmetry
how does the initial reaction of an Asp protease work?
one Asp accepts a proton from an actve site H₂O
which attacks carbonyl carbon of peptide linkage
simultaneously, other Asp donates a proton to oxygen of peptide carbonyl group
what is HIV-1 protease?
asp protease which cleaves polyprotin products of HIV genome
what does function of the HIV-1 protease imitate?
mammalian asp protease function
what is the structure of HIV-1 protease?
99 AAs for virus binding to form heterodimer
activates ability of protease to cleave substrate
simpler than mammalian heterodimer which is genetically economical
what is the life cycle of HIV?
- attachment of virus at CD4 receptor and chemokine co-receptors CXCR4 or CCR5
- viral fusion and uncoating
3-5. HIV reverse transciptase makes single DNA copy of viral RNA and makes another to form double-stranded viral DNA - migration to nucleus
7-8. integration of viral DNA into cellular DNA by enzyme integrase
9-11. transcription and RNA processing
12-13. protein synthesis - protease cleaves polyppetides into functional HI proteins and virion assembles
- virion budding
- virion maturation
what processing is required to produce mature HIV proteins?
the gag-pol polyrpitein must be cleaved by another protease
what can protease inhibitors be used as?
AIDS drugs
HIV therapy
how can HIV-1 protease inhibitors be used to prevent HIV production?
during HIV reproduction cycle, specific protease needed to process GAG and POL polyproteins into mature HIV components
if HIV-1 protease can be selectively inhibited, new HIV particles cannot form
if protease activity missing, non-infectous HIV formed
HIV protease inhibitors specific to HIV protease as it differs from human protease
are any protease inhibitors in use?
several marketed as HIV drugs