Two histidines his 94 and his 96 hca i numbering are

Info icon This preview shows pages 56–59. Sign up to view the full content.

View Full Document Right Arrow Icon
exposed to solvent. Two histidines (His-94 and His-96, HCA I numbering) are bound to zinc via their NE2 atoms, whereas one (His-
Image of page 56

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
50 CA I ....................... ~::? 2 f--_---'--- '-_C_A_I_IT_----i 0. CA II 6 CA II CA I 5 4 <ii CA III ",,'" OJ 3 g 2 pH Figure 2.2 pH dependence of k cat and K m values for CO 2 hydration catalyzed by carbonic anhydrase I, II, and III isoenzymes. 33 ,36 119) is bound via its Nol atom (Figure 2.4). It is quite general that histidines bind zinc equally well by either of the two histidine nitrogens, the preference being probably dictated by the steric constraints imposed by the protein folding. The three histidine NH protons are all engaged in H-bonding (Figure 2.4). His- tidine-119 is involved in H-bonding with a glutamate residue. As mentioned, this could be a way of controlling the basicity of the metal ligands. A solvent molecule bound to zinc is involved in an H-bond with Thr-199, which in tum is H-bonded to Glu-106. This H-bonding network is important for understanding the subtle structural changes that occur with pH changes; these could, in prin- ciple, account for the pH-dependent properties. Although the structure of crys- tals grown at pH 8 in sulfate-containing buffer gives some indication of a single solvent molecule bound to zinc (Figures 2.3 and 2.5 See color plate section, pages C2, C3.), theoretical studies indicate that two water molecules can be at bonding distances. 42 Such a finding is consistent with spectroscopic studies on other derivatives and with the concept that attachment and detachment of sub- strates occur through five coordination. Just as is true for every zinc enzyme in which zinc is at the catalytic site, activity is lost if the metal is removed, and is restored by zinc uptake. The tertiary structure of carbonic anhydrase is maintained in the absence of zinc; even the denatured apoprotein can refold spontaneously from a random coil to a native-like conformation. Although such a process is accelerated by zinc,43,44
Image of page 57
TQYr-~ "-N~ ~ ~ I H 7 ,W---- 0 --:2~~~:~g~ t N 0___ ,0 Thr- I ); 0_-_=:0 ----- 0-- 0'--- 0 199 Thr-200 N C~ I Glu- "N ~~- __ -r~O-!N\\ /'-.- 11Z-/HY J ;\ ~N -r - N\\ //NH --~"HN~ Asn- His- N ~ \;:::::N, Asn-67 244 96 () ~~~ C6"H"-90 ~~S- ~--Z \ // ~ I H /'" NI---O ~ , /' Y ' - Tyr-194 Gln- 8er-29 92 Trp-209 Figure 2.4 Schematic representation of the active site of human carbonic anhydrase II. Hydrogen bonds (---) and ordered water molecules (0) are indicated. 41 the presence of the metal does not seem to be an absolute requirement for the correct folding of CA, whereas it is an absolute requirement for several other metalloproteins. 23 ,29,30 Anions are attracted in the metal cavity by the positive Zn(N 3 0H 2 )2+ moiety, and are believed to bind to zinc in carbonic anhydrase very effectively; so their use should be avoided as much as possible if the goal is to study the enzyme as it is. When the protein is dialyzed against freshly doubly distilled or carefully deionized water under an inert atmosphere, the pH of the sample approaches the isoelectric point, which is below 6 for HCA I and bovine (BCA II) enzymes.
Image of page 58

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Image of page 59
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern