Characterization of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors.
The hydrolyses of 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate and of a new, more soluble, substrate, 4-nitrophenyl 4'-(3-aza-7-hydroxy-2-oxoheptyl)phenyl carbonate, each catalysed by a polyclonal antibody preparation elicited in a sheep by use of an analogous phosphate immunogen, were shown to adhere closely to the Michaelis-Menten equation, in accordance with the growing awareness that polyclonal catalytic antibodies may be much less heterogeneous than had been supposed. The particular value of studies on polyclonal catalytic antibodies is discussed briefly. Both the kcat and kcat/K(m) values were shown to increase with increase in pH across a pKa of approx. 9. Group-selective chemical modification studies established that the side chains of tyrosine and arginine residues are essential for catalytic activity, and provided no evidence for the involvement of side chains of lysine, histidine or cysteine residues. The combination of evidence from the kinetic and chemical modification studies and from studies on the pH-dependence of binding suggests that catalysis involves assistance to the reaction of the substrate with hydroxide ions by hydrogen-bond donation at the reaction centre by tyrosine and arginine side chains. This combination of hydrogen-bond donors appears to be a feature common to a number of other hydrolytic catalytic antibodies. High-pKa acidic side chains may be essential for the effectiveness of catalytic antibodies that utilize hydroxide ions.
Resmini
M
M
Department of Biochemistry, Queen Mary and Westfield College, University of London, U.K.
Vigna
R
R
Simms
C
C
Barber
N J
NJ
Hagi-Pavli
E P
EP
Watts
A B
AB
Verma
C
C
Gallacher
G
G
Brocklehurst
K
K
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D002254:Carbonates / Q000378:metabolism; D002384:Catalysis; D004797:Enzyme-Linked Immunosorbent Assay; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D012756:Sheep / Q000276:immunology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D000906:Antibodies; D002254:Carbonates; D014443:Tyrosine; D001120:Arginine