Speeding Up Proton Transfer in a Fast Enzyme:  Kinetic and Crystallographic Studies on the Effect of Hydrophobic Amino Acid Substitutions in the Active Site of Human Carbonic Anhydrase II†

Biochemistry ◽  
2007 ◽  
Vol 46 (12) ◽  
pp. 3803-3813 ◽  
Author(s):  
S. Zoë Fisher ◽  
Chingkuang Tu ◽  
Deepa Bhatt ◽  
Lakshmanan Govindasamy ◽  
Mavis Agbandje-McKenna ◽  
...  
Keyword(s):  
Amino Acid ◽  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Active Site ◽  
Carbonic Anhydrase Ii ◽  
Amino Acid Substitutions ◽  
Enzyme Kinetic ◽  
Hydrophobic Amino Acid ◽  
Human Carbonic Anhydrase

scholarly journals Effect of Active-site Mutation at Asn67 on the Proton Transfer Mechanism of Human Carbonic Anhydrase II

Biochemistry ◽  
2009 ◽  
Vol 48 (33) ◽  
pp. 7996-8005 ◽  
Author(s):  
C. Mark Maupin ◽  
Jiayin Zheng ◽  
Chingkuang Tu ◽  
Robert McKenna ◽  
David N. Silverman ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Active Site ◽  
Transfer Mechanism ◽  
Carbonic Anhydrase Ii ◽  
Site Mutation ◽  
Human Carbonic Anhydrase

scholarly journals Importance of C-Terminal Knot Structure in Carbonic Anhydrase II (Part I): Role of C-Terminal Knot Forming GLN253 on Enzymatic Property of Human Carbinic II

1970 ◽  
Vol 14 ◽  
pp. 1-9
Author(s):  
Mohammad Taufiq Alam
Keyword(s):  
Amino Acid ◽  
Carbonic Anhydrase ◽  
Point Mutation ◽  
Amino Acid Residue ◽  
Active Site ◽  
Terminal Region ◽  
Carbonic Anhydrase Ii ◽  
C Terminus ◽  
Human Carbonic Anhydrase

In both, bovine and human carbonic anhydrase II, a conserved glutamine residue occupies the position in the middle of the knot, which is formed by intercrossing of C-terminal end with N-terminal region. Previous studies have indicated that C-terminus is not the part of an active site, but truncation of 7 amino acid residue in this region can have marked effects on stability of the enzyme (data not published). To gain further insight into the role of specific amino acid residue in C-terminal region, site directed mutagenesis was used to introduce point mutation. Substitution of glutamine with cysteine was chosen because the cysteine residue is less hydrophilic as compared with glutamine and thus, may disrupt the hydrophilic environment in this region. Result indicates that Gln253 located within the C-terminus knot topology plays a significant role in normal function of the enzyme. Thus, C-terminal region might mediate cooperativity between the central active site of the enzyme through proper formation of knot. Key words: Human carbonic anhydrase II; knot topology; point mutation J. bio-sci. 14: 1-9, 2006

scholarly journals Active-site solvent replenishment observed during human carbonic anhydrase II catalysis

IUCrJ ◽  
2018 ◽  
Vol 5 (1) ◽  
pp. 93-102 ◽  
Author(s):  
Jin Kyun Kim ◽  
Carrie L. Lomelino ◽  
Balendu Sankara Avvaru ◽  
Brian P. Mahon ◽  
Robert McKenna ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Active Site ◽  
Water Reservoir ◽  
Bound Water ◽  
Nucleophilic Attack ◽  
Carbonic Anhydrase Ii ◽  
Intermediate Water ◽  
Water Network ◽  
Human Carbonic Anhydrase

Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO2/HCO3 −. Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO2 pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (WI′) is observed next to the previously observed intermediate water WI, and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH− on CO2. This mechanism explains how the zinc-bound water (WZn) and W1 are replenished, which are directly responsible for the reconnection of the His64-mediated proton-transfer water network. This study provides the first `physical' glimpse of how a water reservoir flows into the hCA II active site during its catalytic activity.

scholarly journals Structural and Kinetic Study of the Extended Active Site for Proton Transfer in Human Carbonic Anhydrase II

Biochemistry ◽  
2010 ◽  
Vol 49 (30) ◽  
pp. 6394-6399 ◽  
Author(s):  
John F. Domsic ◽  
Wilton Williams ◽  
Suzanne Z. Fisher ◽  
Chingkuang Tu ◽  
Mavis Agbandje-McKenna ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Kinetic Study ◽  
Active Site ◽  
Carbonic Anhydrase Ii ◽  
Human Carbonic Anhydrase

scholarly journals Water Networks in Fast Proton Transfer during Catalysis by Human Carbonic Anhydrase II

Biochemistry ◽  
2012 ◽  
Vol 52 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Rose Mikulski ◽  
Dayne West ◽  
Katherine H. Sippel ◽  
Balendu Sankara Avvaru ◽  
Mayank Aggarwal ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Carbonic Anhydrase Ii ◽  
Water Networks ◽  
Fast Proton ◽  
Human Carbonic Anhydrase
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