X-Ray Diffraction Studies of Enzymes

Blow, D. M.; Steitz, T.A.

doi:10.1146/annurev.bi.39.070170.000431

Cited by 201 publications

(71 citation statements)

References 83 publications

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“…There are at least two possible functions for the glucose-induced conformational change: to allow an "embracing mechanism" (30) or to provide specificity (1,24). The stereochemical mechanism of hexokinase may require the enzyme to surround or "embrace" its substrates such that the only way in which the substrates can enter and leave the active site is for the enzyme to open and close (30). The mechanism of hexokinase remains to be established.…”

Section: Discussionmentioning

confidence: 99%

Glucose-induced conformational change in yeast hexokinase.

Bennett¹,

Steitz²

1978

Proc. Natl. Acad. Sci. U.S.A.

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299

127

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Section: Discussionmentioning

confidence: 99%

Glucose-induced conformational change in yeast hexokinase.

Bennett¹,

Steitz²

1978

Proc. Natl. Acad. Sci. U.S.A.

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299

127

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“…However, the stabilization of the tetrahedral intermediate in the oxyanion pocket does seem to be a way in which the structure of the substrate binding site promotes catalysis (8,10,20,26,43 There is complete agreement among the structures of chymotrypsino gen (7,17,56) and the two trypsinogen structural determinations (16,30) that the oxyanion hole, in particular the backbone NH of G1y 193, is not properly oriented to bind the oxyanion of the tetrahedral intermediate. This is due to the reorientation of Asp 194, which interacts with the imidazole of His 40 in the zymogen structures but binds the newly formed a-amino group of lIe 16 in the activated enzyme.…”

Section: On the Role Of Asp 102 In Catalysismentioning

confidence: 99%

Crystallographic and NMR Studies of the Serine Proteases

Steitz¹,

Shulman²

1982

Annu. Rev. Biophys. Bioeng.

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207

117

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Since the serine proteases form the most thoroughly understood family of enzymes, it is of some interest to consider what the two principal physical techniques of studying enzymes, crystallography and nuclear magnetic resonance (NMR), have told us about the details of their enzymatic mechanism. Due to limitations of space, this review cannot be compre hensive nor can it describe the history of the current ideas concerning the action of these enzymes.Two major issues have attracted the attention of both diffraction and NMR studies in the past ten years: the first concerns the nature and the role of the catalytic triad of serine 195, histidine 57, and aspartic acid 102; the second is the possibility of induced distortions upon complexa tion of either the substrate or the enzyme. We first consider these issues in terms of the crystallographic studies and then of the NMR experi ments. Finally, we interpret these results in terms of a detailed structural and electronic mechanism. Our conclusions, subject to further test, are that the triad does not exist in the resting state of the enzyme because the serine-histidine bond is not formed. However, we suggest that this bond does form upon complexation with substrates allowing the triad to play an important role in catalysis.

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“…The peptidases, particularly the so-called serine proteases (chymotrypsin and subtilisin, for instance) have in their active centres a histidine residue whose imidazole reveals a somewhat elevated pK value; also the serine proteases are active at the alkaline side of the pK value (Blow & Steitz, 1970). Similar properties in the structures of the active centres are further suggested by the inactivation of both pantothenase and serine proteases by phenylmethanesulphonyl fluoride.…”

Section: Kinetic Equationmentioning

confidence: 87%

Kinetic studies on pantothenase from Pseudomonas fluorescens. Effects of pH on substrate and inhibitor binding

Airas¹

1976

Biochemical Journal

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The velocity of the pantothenase-catalysed hydrolysis of pantothenate was studied over pH5.5-9, and in the presence of oxalate or oxaloacetate as an inhibitor. The pH-dependence of the reaction can be described by a kinetic equation containing two ionizations of the enzyme, with one ionizable group located at the substrate-binding site, and the other at the inhibitor-binding site. The K,, value of pantothenase to pantothenate depends on the buffer used, and phosphate tends to give somewhat lower values than other buffers. Km also depends on pH, the best activities being observed at basic pH values. The pH-independent Km is 7.6mM in phosphate buffer at 20°C; the corresponding KR " value at pH7 is 15mM. The pK value of the ionizable group at the substrate-binding site was measured by two methods: from the pH-rate profile and from the pH-Km profile. pK is 7.0 in phosphate buffer at 20°C, ranging in various buffers between 6.9 and 7.3. The van't Hoff enthalpies of substrate binding and H+ ion binding were -14kJ/mol and -50kJ/mol respectively. The inhibition by oxalate or oxaloacetate is of non-competitive type and depends on pH, the inhibitors being effective at acidic pH values. The pK value of the ionizable group at the inhibitor-binding site was derived from the measurements of the K, values over the pH range 6-7.5. The pK value was 6.4 in oxaloacetate inhibition, the pH-independent K, being 0.36mM, and the corresponding Ka,' about 1.8mM at pH7. Phenylmethanesulphonyl fluoride was capable of inactivating pantothenase.PantothenasefromPseudomonasfluorescens(pantothenate amidohydrolase, EC 3.5

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X-Ray Diffraction Studies of Enzymes

Cited by 201 publications

References 83 publications

Glucose-induced conformational change in yeast hexokinase.

Glucose-induced conformational change in yeast hexokinase.

Crystallographic and NMR Studies of the Serine Proteases

Kinetic studies on pantothenase from Pseudomonas fluorescens. Effects of pH on substrate and inhibitor binding

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