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Biomedical subjects

R K Gupta

Publications and source records attributed to R K Gupta.

At least 739 records · Page 41Linked to original sources
PubMed1977

A novel nuclear relaxation approach for estimating distance between enzyme- and nucleotide-bound metal ions at the catalytic site of pyruvate kinase.

This communication introduces a nuclear relaxation approach for an estimation of the distance between two paramagnetic metal ion sites on a metal-activated enzyme. The method is based on the existence of an exchange of unpaired electron spin magnetizations between the two metals via energy-conserving concerted mutual spin flips which arise from time-dependent dipolar interactions of the electronic magnetizations. This cross-relaxation of electronic magnetizations depends on the inverse sixth power of the intermetal distance and may, under suitable conditions, affect the longitudinal relaxation rate of inner sphere water protons by altering the electron-proton dipolar correlation time when the latter is dominated by electron spin relaxation. The technique is applied to estimate the distance of 5.2 +/- 0.9 A between Mn2+ and Cr3+ in the pyruvate kinase-Mn2+-ATPCr3+ complex and indicates the existence of a van der Waals contact between the hydration spheres of the enzyme- and nucleotide-bound metal ions.

Adenosine Triphosphate↗
PubMed1977

Specificity of 2-keto-3-deoxygluconate-6-P aldolase for open chain form of 2-keto-3-deoxygluconate-6-P.

2-Keto-3-deoxygluconate-6-P exists as an euqilibrium of three forms at 25 degrees measurable by 13C NMR: alpha-furanose anomer (41%), beta-furanose anomer (50%), and open chain keto (9%). The three forms are interconverted rapidly (greater than 0.5 s-1) so that the unidirectional rates of furanose ring opening and closing can be quantitated by an NMR line broadening method. The 2-keto-3-deoxygluconate aldolase is specific for only one of these forms, the open chain keto form. The rates for ring opening calculated from the rapid kinetic enzyme system compare closely with those obtained with the NMR method.

Aldehyde-Lyases↗
PubMed1976

Nuclear relaxation and gelation study of the interaction of organophosphates with human normal and sickle hemoglobins. In vitro gelation of sickle oxyhemoglobin in the presence of inositol hexaphosphate.

31P relaxation studies reveal a 3-fold enhancement of the longitudinal relaxation rate of both phosphoryl groups of hemoglobin-bound 2,3-bisphosphoglycerate upon conversion of methemoglobin to fluoromethemoglobin presumably due to an order of magnitude increase in the electron spin relaxation time. The enhancement of the longitudinal components of 31P relaxation (T(-1)1pr) upon binding to hemoglobin is not exchange-limited, since it is more than an order of magnitude smaller than the effect observed on the transverse components (T(-1)2pr). From the observed paramagnetic component, T(-1)1M, of the bound state relaxation rate of 2,3-bisphosphoglycerate, using the correlation time tau s obtained from the frequency dependence of water proton relaxation, we obtained an NMR root mean sixth distance from the four heme iron atoms to each of the 31P nuclei of 24 +/- 1 A. This is in excellent agreement with the x-ray crystallographic determination of this distance of (25 +/- 1) A in the 2,3-bisphosphoglycerate-deoxyhemoglobin complex, indicating that the spatial disposition of the allosteric site in the deoxy and oxy conformations of hemoglobin relative to the various heme irons may be the same, and that the same protein groups may be involved in binding 2,3-bisphosphoglycerate to the two forms of hemoglobin. Water proton relaxation studies reveal the existence of different conformational states of methemoglobins with 2,3-bisphosphoglycerate and inositol hexaphosphate. Inositol hexaphosphate alters the conformation to a strained (T) state with deoxy-like quaternary and tertiary globin structure as indicated by the finding that equimolar amounts of inositol heasphosphate induce gelation in a 4 mM sickle methemoglobin solution at temperatures greater than or equal 24 degrees. More interestingly, oxyhemoglobin S shows an identical thermodynamically reversible gelation behavior, with the same transition temperature (24 degrees), arguing against a mutual coupling of the protein conformation and the heme spin state in functional ferrohemoglobins. High ionic strengths (approximately 1 M) and pH values above neutrality block inositol hexaphosphate induced gelation of sickle met- and oxyhemoglobins. Unlike inositol hexaphosphate, the presence of saturating amounts of 2,3-bisphosphoglycerate does not promote gelation of a 4 mM met- or oxyhemoglobin S solution.

Diphosphoglyceric Acids↗
PubMed1976

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.

Adenosine Triphosphate↗