scholarly journals Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin

1984 ◽  
Vol 220 (2) ◽  
pp. 405-416 ◽  
Author(s):  
W Kullmann
Keyword(s):  
Peptide Synthesis ◽  
Initial Velocity ◽  
Substrate Inhibition ◽  
Peptide Bond ◽  
Ethyl Esters ◽  
Acyl Donor ◽  
Peptide Bond Formation ◽  
Ping Pong ◽  
Acyl Acceptors ◽  
Acyl Donors

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. N alpha-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of alpha-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin- and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.

scholarly journals Purification and steady-state kinetics of adenosine 5′-pyrophosphate sulphurylase from baker's yeast

1977 ◽  
Vol 165 (1) ◽  
pp. 149-155 ◽  
Author(s):  
R G Nicholls
Keyword(s):  
Exchange Reaction ◽  
Initial Velocity ◽  
Isotope Exchange ◽  
Substrate Inhibition ◽  
Deae Cellulose ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Kinetics Of

ADP sulphurylase (EC 2.7.7.5) was purified by chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was assayed by measuring the incorporation of [32P]Pi into ADP in the presence of the substrate for the reverse reaction, adenosine 5′-sulphatophosphate. In the concentration ranges investigated, by using initial-velocity, product-inhibition and isotope-exchange studies, the data were consistent with a Ping Pong reaction mechanism, with Km for adenosine 5′-sulphatophosphate of 1.20 +/- 0.08 mM and a Km for Pi of 4.95 +/- 0.15 mM. Competitive substrate inhibition by Pi (Ki = 11.7 +/- 0.3 mM) was found. ADP sulphurylase catalyses a sulphate-independent Pi-ADP exchange reaction, the kinetics of which are consistent with the kinetics of the overall reaction, inconsistent with the assay of Burnell & Anderson [(1973) Biochem. J. 133, 417-428], which is based on a sulphate-dependent Pi-ADP exchange reaction.

On the Occurrence of Monoacylglycerol Derivatives in Lipid Metabolism of Chloroplasts

1982 ◽  
Vol 37 (3-4) ◽  
pp. 218-225 ◽  
Author(s):  
Andreas Sauer ◽  
Klaus-Peter Heise
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipid Fraction ◽  
Acyl Donor ◽  
Rapid Synthesis ◽  
Acid Incorporation ◽  
Fatty Acid Incorporation ◽  
Acyl Acceptors ◽  
Fatty Acid Species ◽  
Acyl Donors

Abstract The aim of this investigation was to elucidate the rapid synthesis o f monogalactosyl mono­ glycerides and their role as intermediary acyl-acceptors in galactolipid synthesis of spinach chlo­ roplasts. The problem was attacked by studying the incorporation o f sn-[14C]glycerol-3-phosphate and [l-14C]acetate into the lipid fraction o f gently shocked and reconstituted preparations. The data revealed: 1 . a concurrent accumulation o f both monoglycerides and monogalactosyl monoglycerides under acidic incubation conditions, with C16-fatty acid species predominating, 2 . similarities in the fatty acid incorporation of both monoacyllipids, 3. the occurrence o f two isomeric forms viz. 1 -and 2-O-acyl-isomers o f these lipids. Thus, it appears that monogalactosyl monoglycerides are synthesized by galactosylation o f mo­ noglycerides rather than by galactolipase hydrolysis. Both monoacyllipids are likely to be derived from the corresponding lysophosphatidic acids by dephosphorylation. Their fatty acid incorporation pattern therefore may contribute to an under­ standing of the specific esterification o f different fatty acids at the Ct-and exp osition of the gly­ cerol moiety of galactolipids. Analysis o f the specific acylation o f monoglycerides and monogalac­ tosyl monoglycerides as well as the nature of the acyl donors involved in this fatty acid transfer yielded the following observations: 5. The position of the fatty acids within the monoacyllipids seems to depend on whether acyl-ACP or acyl-CoA is the primary acyl donor. 6 . The characteristics of the fatty acid incorporation into monoglycerides and their galactosylated derivatives support the notion that a successive acylation o f sn-glycerol-3-phosphate occurs first in the C2-and then in the Q-position. 7. In contrast, the chain length o f the fatty acids incorporated seems to be determined by such fac­ tors as pH and the concentration o f sn-glycerol-3-phosphate. This observation suggests that these parameters may act by controlling the elongation o f ACP-bound C16-fatty acids to their C18-species.

Role of the Small (40S) Subunits in the Structure of the Interface of Eukaryotic Ribosomes

1980 ◽  
Vol 38 ◽  
pp. 548-549
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Structural Similarity ◽  
High Resolution Electron Microscopy ◽  
Peptide Bond ◽  
Uranyl Acetate ◽  
Mutual Orientation ◽  
Mutual Position ◽  
Peptide Bond Formation ◽  
Resolution Electron ◽  
Direct Technique ◽  
Structural Details

Ribosomes are ribonucleoprotein particles which process the genetic information coded in mRNA into protein synthesis. The analogy in function and composition of ribosomes from various sources, both prokaryotic and eukaryo-tic, imply a structural similarity. At present, high resolution electron microscopy is the most direct technique with a potential to resolve the extent of the structural homology of ribosomal particles at a macromolecular level. The structure of ribosomes is highly complex as a result of the large number of their constituents. In general, 80S eukaryotic monosomes consist of two uneven subunits - large (60S) and small (40S) - accomodating four different RNAs and approximately 80 different proteins. Mutual orientation of both subunits on the monosome is of particular interest because it determines the interface, the supposed site of interactions of ribosomes with other macro-molecules involved in peptide bond formation. Since entrapping of the contrasting solution (0.5% aqueous uranyl acetate) obscures all structural details in the interface, information on its architecture is limited to an indirect reconstruction based on the established 3-D structure of both sub-units and their mutual position after association.

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